Insulin-like growth factor agonist molecules

ABSTRACT

Compounds are provided that prevent the interaction of an IGF with any one of its binding proteins and not to a human IGF receptor. These IGF agonist compounds, which include peptides, are useful to increase serum and tissue levels of active IGFs in a mammal

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention relates to molecules useful as agonists of theinsulin-like growth factors (IGFs). More particularly, these moleculesprevent the interaction of an IGF with one or more of its IGF bindingproteins. Such molecules can be used, for example, in any methods wherethe IGFs are used, for example, in treating hyperglycemic,obesity-related, neurological, cardiac, renal, immunologic, and anabolicdisorders.

2. Description of Background and Related Art

There is a large body of literature on the actions and activities ofIGFs (IGF-I, IGF-II, and IGF variants). Human IGF-I is a 7649-daltonpolypeptide with a pI of 8.4 (Rinderknecht and Humbel, Proc. Natl. Acad.Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253:2769 (1978)) belonging to a family of somatomedins with insulin-like andmitogenic biological activities that modulate the action of growthhormone (GH). Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974);Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, HandbookExp. D Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem., 25: 49(1986); U.S. Pat. No. 4,988,675; WO 91/03253; WO 93/23071.

Like GH, IGF-I is a potent anabolic protein. See Tanner et al., ActaEndocrinol., 84: 681-696 (1977); Uthne et al., J. Clin. Endocrinol.Metab., 39: 548-554 (1974). See also Ross et al., Intensive Care Med.,19 Suppl. 2: S54-57 (1993), which is a review of the role of insulin,GH, and IGF-I as anabolic agents in the critically ill. IGF-I hashypoglycemic effects similar to those of insulin, but also promotespositive nitrogen balance. Underwood et al., Hormone Res., 24: 166(1986); Guler et al., N. Engl. J. Med., 317: 137 (1987). Due to thisrange of activities, IGF-I is being tested in humans for such widelydisparate uses as wound healing, treatment of diabetes, reversal ofwhole body catabolic states, treatment of heart conditions such ascongestive heart failure, and treatment of neurological disorders. Guleret al., Proc. Natl. Acad. Sci. USA, 85: 4889-4893 (1988); Duerr et al.,J. Clin. Invest., 95: 619-627 (1995); and Science, 264: 772-774 (1994).

U.S. Pat. Nos. 5,273,961; 5,466,670; 5,126,324; 5,187,151; 5,202,119;5,374,620; 5,106,832; 4,988,675; 5,106,832; 5,068,224; 5,093,317;5,569,648; and 4,876,242; WO 92/11865; WO 96/01124; WO 91/03253; WO93/25219; WO 93/08826; and WO 94/16722 disclose various methods oftreating mammals, especially human patients, using IGF-I. In addition,clinical uses of IGF-I are described, for example, in Bondy, Ann Intern.Med., 120: 593-601 (1994).

As one specific use, IGF-I has been found to exert a variety of actionsin the kidney. Hammerman and Miller, Am. J. Physiol., 265: F1-F14(1993). It has been recognized for decades that the increase in kidneysize observed in patients with acromegaly is accompanied by asignificant enhancement of glomerular filtration rate. O'Shea andLayish, J. Am. Soc. Nephrol., 3: 157-161 (1992). U.S. Pat. No. 5,273,961discloses a method for prophylactic treatment of mammals at risk foracute renal failure. In humans IGF-I has been shown to preserve renalfunction post-operatively. Franklin et al., Am. J. Physiol., 272:F257-F259 (1997). Infusion of the peptide in humans with normal renalfunction increases glomerular filtration rate and renal plasma flow.Guler et al., Acta Endocrinol., 121: 101-106 (1989); Guler et al., Proc.Natl. Acad. Sci. USA, 86: 2868-2872 (1989); Hirschberg et al., KidneyInt., 43: 387-397 (1993); U.S. Pat. No. 5,106,832. Further, humans withmoderately reduced renal function respond to short-term (four days)IGF-I administration by increasing their rates of glomerular filtrationand renal plasma flow. Hence, IGF-I is a potential therapeutic agent inthe setting of chronic renal failure. O'Shea et al., Am. J. Physiol.,264: F917-F922 (1993).

Despite the fact that IGF-I can enhance renal function for thoseexperiencing end-stage chronic renal failure, the enhancements of theglomerular filtration rate and renal plasma flow induced by IGF-Ishort-term do not persist during long-term administration and incidenceof side-effects is high. Miller et al., Kidney International, 46:201-207 (1994).

For complete reviews of the effect of IGF-I on the kidney, see, e.g.,Hammerman and Miller, Am. J. Physiol., 265: F1-F14 (1993) and Hammermanand Miller, J. Am. Soc. Nephrol., 5: 1-11 (1994).

As to anabolic indications for IGF-I, in HIV-infected patients treatedconsecutively with IGF-I, the IGF-I promoted anabolism, buttachyphylaxis developed rapidly in the patients. Lieberman et al., U.S.Endocrine Meeting, June 1993 (Abst. 1664); Lieberman et al., J. Clin.Endo. Metab., 78: 404-410 (1994). In patients with severe head injuries,a condition associated with profound hypercatabolism and nitrogen loss,infusion of IGF-I produced only a transient positive nitrogen balance.In the first week the patients experienced a positive nitrogen balance,but during the second week, a negative nitrogen balance developed. Chenet al., U.S. Endocrine Meeting, June 1993 (Abst. 1596).

IGF-I has hypoglycemic effects in humans similar to those of insulinwhen administered by intravenous bolus injection. Underwood et al.,Hormone Research, 24: 166 (1986). IGF-I is known to exertglucose-lowering effects in both normal (Guler et l., N. Engl. J. Med.,supra) and diabetic individuals (Schoenle et al., Diabetologia, 34:675-679 (1991); Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992);Sherwin et al., Hormone Research, 41 (Suppl. 2): 97-101 (1994); Takanoet al., Endocrinol. Japan, 37: 309-317 (1990); Guler et al., ActaPaediatr. Scand. (Suppil.), 367: 52-54 (1990)), with a time coursedescribed as resembling regular insulin. See also Kerr et al., "Effectof Insulin-like Growth Factor 1 on the responses to and recognition ofhypoglycemia," American Diabetes Association (ADA), 52nd Annual Meeting,San Antonio, Tex., Jun. 20-23, 1992, which reported an increasedhypoglycemia awareness following recombinant human IGF-I (rhIGF-I)administration. In addition, single administration of rhIGF-I reducesovernight GH levels and insulin requirements in adolescents with IDDM.Cheetham et al., Clin. Endocrinol., 40: 515-555 (1994); Cheetham et al.,Diabetologia, 36: 678-681 (1993).

The administration of rhIGF-I to Type II diabetics, as reported bySchalch et al., J. Clin. Metab., 77: 1563-1568 (1993), demonstrated afall in both serum insulin as well as a paralleled decrease in C peptidelevels. This indicated a reduction in pancreatic insulin secretion afterfive days of IGF-I treatment. This effect has been independentlyconfirmed by Froesch et al., Horm. Res., 42: 66-71 (1994). In vivostudies in normal rats also illustrate that IGF-I infusion inhibitspancreatic insulin release. Fursinn et al., Endocrinology, 135:2144-2149 (1994). In addition, in pancreas perfusion preparations, IGF-Ialso suppressed insulin secretion. Leahy et al., Endocrinology, 126:1593-1598 (1990). Despite these clear in vivo inhibitory effects ofIGF-I on insulin secretion in humans and animals, in vitro studies havenot yielded such uniform results.

RhIGF-I has the ability to improve insulin sensitivity. For example,rhIGF-I (70 μg/kg bid) improved insulin sensitivity in non-diabetic,insulin-resistant patients with myotonic dystrophy. Vlachopapadopoulouet al., J. Clin. Endo. Metab., 12: 3715-3723 (1995). Saad et al.,Diabetologia, 37: Abstract 40 (1994) reported dose-dependentimprovements in insulin sensitivity in adults with obesity and impairedglucose tolerance following 15 days of rhIGF-I treatment (25 μg and 100μg/kg bid). RhIGF-I also improved insulin sensitivity and glycemiccontrol in some patients with severe type A insulin resistance (Schoenleet al., Diabetologia, 34: 675-679 (1991); Morrow et al., Diabetes, 42(Suppl.): 269 (1993) (abstract); Kuzuya et al., Diabetes, 42: 696-705(1993)) and in other patients with non-insulin dependent diabetesmellitus. Schalch et al., "Short-term metabolic effects of recombinanthuman insulin-like growth factor I (rhIGF-I) in type II diabetesmellitus", in: Spencer EM, ed., Modern Concepts of Insulin-like GrowthFactors (New York: Elsevier: 1991) pp. 705-715; Zenobi et al., J. Clin.Invest., 90: 2234-2241 (1993).

A general scheme for the etiology of some clinical phenotypes that giverise to insulin resistance and the possible effects of administration ofIGF-I on selected representative subjects is given in several referencesSee, e.g., Elahi et al., "Hemodynamic and metabolic responses to humaninsulin-like growth factor-i (IGF-I) in men," in: Modern Concepts ofInsulin-Like Growth Factors, (Spencer, EM, ed.), Elsevier, N.Y., pp.219-224 (1991); Quinn et al., New Engl. J. Med., 323: 1425-1426 (1990);Schalch et al., "Short-term metabolic effects of recombinant humaninsulin-like growth factor 1 (rhIGF-I) in type 11 diabetes mellitus,"in: Modern Concepts of Insulin-Like Growth Factors, (Spencer, EM, ed.),Elsevier, N.Y., pp. 705-714 (1991); Schoenle et al., Diabetologia, 34:675-679 (1991); Usala et al., N. Eng. J. Med., 327: 853-857 (1992);Lieberman et al., J. Clin. Endo. Metab., 75: 30-36 (1992); Zenobi etal., J. Clin. Invest., 90: 2234-2241 (1992); Zenobi et al., J. Clin.Invest., 89: 1908-1913 (1992); Kerr et al., J. Clin. Invest., 91:141-147 (1993). When IGF-I was used to treat Type II diabetic patientsin the clinic at a dose of 120-160 μg/kg twice daily, the side effectsoutweighed the benefit of the treatment. Jabri et al., Diabetes, 43:369-374 (1994). See also Wilton, Acta Paediatr., 383: 137-141 (1992)regarding side effects observed upon treatment of patients with IGF-I.

The IGF binding proteins (IGFBPs) are a family of at least six proteins(Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995); Bach and Rechler,Diabetes Reviews, 3: 38-61 (1995)), with other related proteins alsopossibly binding the IGFs. The IGFBPs bind IGF-I and IGF-II with varyingaffinities and specificities. Jones and Clemmons, supra; Bach andRechler, supra. For example, IGFBP-3 binds IGF-I and IGF-II with asimilar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-II with a muchhigher affinity than they bind IGF-I. Bach and Rechler, supra; Oh etal., Endocrinology, 132, 1337-1344 (1993).

Unlike most other growth factors, the IGFs are present in highconcentrations in the circulation, but only a small fraction of the IGFsis not protein bound. For example, it is generally known that in humansor rodents, less than 1% of the IGFs in blood is in a "free" or unboundform. Juul et al., Clin. Endocrinol., 44: 515-523 (1996); Hizuka et al.,Growth Regulation, 1: 51-55 (1991); Hasegawa et al., J. Clin.Endocrinol. Metab., 80: 3284-3286 (1995). The overwhelming majority ofthe IGFs in blood circulate as part of a non-covalently associatedternary complex composed of IGF-I or IGF-II, IGFBP-3, and a largeprotein termed the acid-labile subunit (ALS). This complex is composedof equimolar amounts of each of the three components. The ternarycomplex of an IGF, IGFBP-3, and ALS has a molecular weight ofapproximately 150,000 daltons, and it has been suggested that thefunction of this complex in the circulation may be to serve as areservoir and buffer for IGF-I and IGF-II, preventing rapid changes infree IGF-I or IGF-II.

IGF-I naturally occurs in human body fluids, for example, blood andhuman cerebral spinal fluid. Although IGF-I is produced in many tissues,most circulating IGF-I is believed to be synthesized in the liver. TheIGFBPs are believed to modulate the biological activity of IGF-I (Jonesand Clemmons, supra), with IGFBP-1 (Lee et al., Proc. Soc. Exp. Biol. &Med., 204: 4-29 (1993)) being implicated as the primary binding proteininvolved in glucose metabolism. Baxter, "Physiological roles of IGFbinding proteins", in: Spencer (Ed.), Modern Concepts of Insulin-likeGrowth Factors (Elsevier, N.Y., 1991), pp. 371-380. IGFBP-1 productionby the liver is regulated by nutritional status, with insulin directlysuppressing its production. Suikkari et al., J. Clin. Endocrinol.Metab., 66: 266-272 (1988).

The function of IGFBP-1 in vivo is poorly understood. The administrationof purified human IGFBP-1 to rats has been shown to cause an acute, butsmall, increase in blood glucose. Lewitt et al., Endocrinology, 129:2254-2256 (1991). The regulation of IGFBP-1 is somewhat betterunderstood. It has been proposed (Lewitt and Baxter, Mol. CellEndocrinology, 79: 147-152 (1991)) that when blood glucose rises andinsulin is secreted, IGFBP-1 is suppressed, allowing a slow increase in"free" IGF-I levels that might assist insulin action on glucosetransport. Such a scenario places the function of IGFBP-1 as a directregulator of blood glucose.

The IGF system is also composed of membrane-bound receptors for IGF-I,IGF-II, and insulin. The Type 1 IGF receptor is closely related to theinsulin receptor in structure and shares some of its signaling pathways.Jones and Clemmons, supra. The IGF-II receptor is a clearance receptorthat appears not to transmit an intracellular signal. Jones andClemmons, supra. Since IGF-I and IGF-II bind to the Type 1 IGF-Ireceptor with a much higher affinity than to the insulin receptor, it ismost likely that most of the effects of IGF-I and IGF-II are mediated bythe Type 1 IGF receptor. Ballard et al., "Does IGF-I ever act throughthe insulin receptor?", in Baxter et al. (Eds.), The Insulin-Like GrowthFactors and Their Regulatory Proteins, (Amsterdam: Elsevier, 1994), pp.131-138.

There has been much work identifying the domains on IGF-I and IGF-IIthat bind to the IGFBPs. Bayne et al., J. Biol. Chem., 265: 15648-15652(1990); U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828. For example, ithas been discovered that the N-terminal region of IGF-I and IGF-II iscritical for binding to the IGFBPs. U.S. Pat. Nos. 5,077,276; 5,164,370;5,470,828. Thus, the natural IGF-I variant, designated des(1-3)IGF-I,binds poorly to IGFBPs.

A similar amount of research has been devoted to identifying the domainson IGF-I and IGF-II that bind to the Type 1 IGF receptor. Bayne et al.,supra; Oh et al., supra. It was found that the tyrosine residues inIGF-I at positions 24, 31, and 60 are crucial to the binding of IGF-I tothe Type 1 IGF receptor. Bayne et al., supra. Mutant IGF-I moleculeswhere one or more of these tyrosine residues are substituted showedprogressively reduced binding to Type 1 IGF receptors. Bayne et al.,supra, also investigated whether such mutants of IGF-I could bind to theType 1 IGF receptor and to the IGFBPs. They found that quite differentresidues on IGF-I and IGF-II are used to bind to the IGFBPs from thoseused to bind to the Type 1 IGF receptor. It is therefore possible toproduce IGF variants that show reduced binding to the IGFBPs, but,because they bind well to the Type 1 IGF receptor, show maintainedactivity in in vitro activity assays.

Also reported was an IGF variant that binds to IGFBPs but not to IGFreceptors and therefore shows reduced activity in in vitro activityassays. Bar et al., Endocrinology, 127: 3243-3245 (1990). In thisvariant, designated (1-27,gly⁴,38-70)-hIGF-I, residues 28-37 of the Cregion of human IGF-I are replaced by a four-residue glycine bridge. Baret al. studied the transport of the mutant IGF-I when it was perfused asa complex with IGFBP through the heart in terms of the localization ofIGFBPs bound to the mutant IGF or to IGF itself. There were no datasupplied by Bar et al. on the localization of the IGF mutant givenalone, only data on the localization of the complex of the IGF mutantand IGFBP. Further, Bar et al. provided no data on any biological orefficacy response to the administration of the IGF mutant.

Other truncated IGF-I variants are disclosed. For example, in the patentliterature, WO 96/33216 describes a truncated variant having residues1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-Isuperagonists which are derivatives of the naturally occurringsingle-chain IGF-I having an abbreviated C domain. The IGF-I analogs areof the formula: BC^(n),A wherein B is the B domain of IGF-I or afunctional analog thereof, C is the C domain of IGF-I or a functionalanalog thereof, n is the number of amino acids in the C domain and isfrom about 6 to about 12, and A is the A domain of IGF-I or a functionalanalog thereof.

Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988)discloses four mutants of IGF-I, three of which have reduced affinity tothe Type 1 IGF receptor. These mutants are: (Phe²³,Phe²⁴,Tyr²⁵)IGF-I(which is equipotent to human IGF-I in its affinity to the Types 1 and 2IGF and insulin receptors), (Leu²⁴)IGF-I and (Ser²⁴)IGF-I (which have alower affinity than IGF-I to the human placental Type 1 IGF receptor,the placental insulin receptor, and the Type 1 IGF receptor of rat andmouse cells), and desoctapeptide (Leu²⁴)IGF-I (in which the loss ofaromaticity at position 24 is combined with the deletion of thecarboxyl-terminal D region of hIGF-I, which has lower affinity than(Leu²⁴)IGF-I for the Type 1 receptor and higher affinity for the insulinreceptor). These four mutants have normal affinities for human serumbinding proteins.

Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988) discloses fourstructural analogs of human IGF-I: a B-chain mutant in which the first16 amino acids of IGF-I were replaced with the first 17 amino acids ofthe B-chain of insulin, (Gln³,Ala⁴)IGF-I, (Tyr¹⁵,Leu¹⁶)IGF-I, and(Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶)IGF-I. These studies identify some of the domainsof IGF-I that are responsible for maintaining high-affinity binding withthe serum binding protein and the Type 2 IGF receptor.

Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses threestructural analogs of IGF-I: (1-62)IGF-I, which lacks thecarboxyl-terminal 8-amino-acid D region of IGF-I;(1-27,Gly⁴,38-70)IGF-I, in which residues 28-37 of the C region of IGF-Iare replaced by a four-residue glycine bridge; and(1-27,Gly⁴,38-62)IGF-I, with a C region glycine replacement and a Dregion deletion.

Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses threeIGF-I analogs in which specific residues in the A region of IGF-I arereplaced with the corresponding residues in the A chain of insulin. Theanalogs are: (Ile⁴¹, Glu⁴⁵, Gln⁴⁶, Thr⁴⁹, Ser⁵⁰,Ile⁵¹, Ser⁵³, Tyr⁵⁵,Gln⁵⁶)IGF-I, an A chain mutant in which residue 41 is changed fromthreonine to isoleucine and residues 42-56 of the A region are replaced;(Thr⁴⁹,Ser⁵⁰,Ile⁵¹)IGF-I; and (Tyr⁵⁵, Gln⁵⁶)IGF-I.

Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990) discloses useof IGF-I analogs that have reduced binding affinity for either the Type1 IGF receptor or binding proteins to study the ligand specificity ofIGFBP-1 and the role of IGFBP-1 in modulating the biological activity ofIGF-I.

WO 94/04569 discloses a specific binding molecule, other than a naturalIGFBP, that is capable of binding to IGF-I and can enhance thebiological activity of IGF-I.

U.S. Pat. Nos. 5,593,844 and 5,210,017 disclose a ligand-mediatedimmunofunctional binding protein assay method that can be used toquantitate the amount of GH binding protein or IGFBP in a liquid sampleby the use of antibodies, where complex formation takes place betweenone of these binding proteins and the hormone ligand that binds to it.

The direction of research into IGF variants has mostly been to make IGFvariants that do not bind to the IGFBPs but show maintained binding tothe IGF receptor. The idea behind the study of such molecules is thatthe major actions of the IGFBPs are proposed to be an inhibition of theactivity of the IGFs. Chief among these variants is the naturalmolecule, des(1-3)IGF-I, which shows selectively reduced affinity forsome of the IGF binding proteins, yet a maintained affinity for the IGFreceptor. U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828, supra.

There is a need in the art for a molecule that acts as an IGF agonist,and also for a molecule that binds to IGF binding proteins with highaffinity and specificity for therapeutic or diagnostic purposes.

SUMMARY OF THE INVENTION

This invention relates to a novel method for providing releasing factorswhich, as part of their actions, prevent binding of an IGF to an IGFBPsuch as by binding to an IGFBP to agonize the action of IGF.Accordingly, the present invention provides a compound that prevents theinteraction of an IGF with any one of its IGFBPs and does not bind to ahuman IGF receptor, excluding (1-27,gly⁴, 38-70)-hIGF-I, excludingantibodies against an IGFBP that do not bind to a human IGF receptor,excluding antibodies that bind to an IGF, and excluding peptides havingthe native sequence of human IGF-I with the tyrosine residues atpositions 24, 31, and/or 60 replaced or deleted.

Preferably, the compound herein binds to an IGFBP, preferably a serumIGFBP. Also, preferably, the compound reduces plasma insulin secretion,reduces plasma GH, and/or reduces blood glucose levels in a mammal.

In other preferred embodiments, the compound herein is a peptide,especially a peptide having about 10 to about 25 amino acid residues,and/or having a cysteine residue at position 5, 6, 7, or 8 numbered fromits N-terminus or having a cysteine residue at position 5, 6, 7, or 8numbered from its C-terminus, or both such cysteine residues, or acysteine residue at position 2 numbered from its N-terminus.

In another embodiment, the invention provides a peptide comprising anamino acid sequence selected from the group consisting of the followingpeptides:

    __________________________________________________________________________    BP3-23      ELDGWVCIKVGEQNLCYLAEG                                                                         (SEQ ID NO: 1),                                     BP3-24 WFKTVCYEWEDEVQCYTLEEG (SEQ ID NO: 2),                                  BP3-25 RVGAYISCSETECWVEDLLDG (SEQ ID NO: 3),                                  BP3-4D3.11 (BP14) VAWEVCWDRHDQGYICTTDS (SEQ ID NO: 4),                        BP3-4D3.11DEL AWEVCWDRHQGYICTTDS (SEQ ID NO: 5),                              BP13 CWDRHDQGYICTTDS (SEQ ID NO: 6),                                          BP3-4B3.3 EESECFEGPGYVICGLVG (SEQ ID NO: 7),                                  BP3-02-OX DMGVCADGPWMYVCEWTE (SEQ ID NO: 8),                                  BP3-01-OX SEEVCWPVAEWYLCNMWG (SEQ ID NO: 9),                                  BP15 SEEVCWPVAEWYLCN (SEQ ID NO: 10),                                         BP16 VCWPVAEWYLCNMWG (SEQ ID NO: 11),                                         BP17 VCWPVAEWYLCN (SEQ ID NO: 12),                                            BP06 TGVDCQCGPVHCVCMDWA (SEQ ID NO: 13),                                      BP08 TVANCDCYMPLCLCYDSD (SEQ ID NO: 14),                                      bp1-01 CRAGPLQWLCEKYFG (SEQ ID NO: 15), and                                   bp1-02 SEVGCRAGPLQWLCEKYFG (SEQ ID NO: 16).                                 __________________________________________________________________________

Also provided herein is a composition comprising one of the compounds orpeptides described above in a pharmaceutically acceptable carrier.Preferably, this composition is sterile.

Uses of these compounds and peptides include all uses that liberate orenhance at least one biological activity of exogenous or endogenousIGFs. They can be used in treating or preventing conditions in which anIGF such as IGF-I is useful, as described below.

Additionally provided herein is a method for increasing serum and tissuelevels of biologically active IGF in a mammal comprising administeringto the mammal an effective amount of a compound that prevents theinteraction of an IGF with any one of its IGFBPs and does not bind ahuman IGF receptor. Preferably, this compound also reduces plasmainsulin secretion, plasma GH secretion, or blood glucose levels in amammal, and does not directly stimulate the secretion or release ofendogenous GH from any species. In other preferred embodiments, thiscompound binds to an IGFBP, such as IGFBP-1 and/or IGFBP-3, and/or doesnot bind to a human Type 1 IGF-I receptor. In addition, the mammal ispreferably human and the compound is preferably a peptide, morepreferably one having about 10 to about 25 amino acid residues. Alsopreferred is where administering the compound, preferably in an amounteffective to produce body weight gain, causes an increase in anabolismin the mammal. Additionally preferred is that glycemic control iseffected in the mammal after the compound is administered.

Isolated nucleic acid encoding the compound herein, if it is a peptide,is also provided, and may be used for in vivo or ex vivo gene therapy.

The compound herein can be administered alone or together with anotheragent such as GH, a GH releasing peptide (GHRP), a GH releasing factor(GHRF), a GH releasing hormone (GHRH), a GH secretagogue, an IGF, an IGFin combination with an IGFBP, an IGFBP, GH in combination with a GHbinding protein (GHBP), insulin, or a hypoglycemic agent (which includesin the definition below an insulin-sensitizing agent such asthiazolidinedione).

In yet another aspect of the invention, a method is provided foreffecting glycemic control in a mammal comprising administering to themammal an effective amount of a compound that prevents the interactionof an IGF with any one of its IGFBPs and does not bind a human IGFreceptor. Preferably, the compound also reduces plasma insulin secretionand blood glucose levels in a mammal and binds an IGFBP. Alsopreferably, the mammal has a hyperglycemic disorder such as diabetes.This method can additionally comprise administering to the mammal aneffective amount of a hypoglycemic agent or insulin.

Also provided is a method for increasing serum and tissue levels ofbiologically active IGF in a mammal, or a method for increasinganabolism in a mammal, or a method for controlling glycemia in a mammalcomprising administering to the mammal an effective amount of thecomposition containing the compound herein.

In another embodiment, a method is provided for determining appropriatedosing of a compound that prevents the interaction of an IGF with anyone of its IGFBPs and does not bind to a human IGF receptor comprising:

(a) measuring the level of an IGF in a body fluid;

(b) contacting the fluid with the compound herein using single ormultiple doses; and

(c) re-measuring the level of an IGF in the fluid, wherein if the fluidIGF level has fallen by an amount sufficient to produce the desiredefficacy for which the compound is to be administered, then the dose ofthe compound is adjustable or adjusted to produce maximal efficacy.

In yet another embodiment, a method is provided for determining theamount of a particular IGFBP or the amount of the compound bound to aparticular IGFBP in a biological fluid so that dosing of the compoundcan be adjusted appropriately. This method involves:

(a) contacting the fluid with 1) a first antibody attached to asolid-phase carrier, wherein the first antibody is specific for epitopeson the IGFBP such that in the presence of antibody the IGF binding sitesremain available on the IGFBP for binding to the compound, therebyforming a complex between the first antibody and the IGFBP; and 2) theabove-identified compound for a period of time sufficient to saturateall available IGF binding sites on the IGFBP, thereby forming asaturated complex;

(b) contacting the saturated complex with a detectably labeled secondantibody which is specific for epitopes on the compound which areavailable for binding when the compound is bound to the IGFBP; and

(c) quantitatively analyzing the amount of the labeled second antibodybound as a measure of the IGFBP in the biological fluid, and thereforeas a measure of the amount of the compound bound.

Also contemplated herein is a kit comprising a container containing apharmaceutical composition containing the compound herein andinstructions directing the user to utilize the composition. This kit mayoptionally further comprise a container containing a GH, a GHRP, a GHRF,a GHRH, a GH secretagogue, an IGF, an IGF complexed to an IGFBP, anIGFBP, a GH complexed with a GHBP, insulin, or a hypoglycemic agent.

Also included herein is a method for predicting the relative affinityfor binding to a ligand of a peptide that competes with a polypeptidefor binding to the ligand, which peptide is derived from aphage-displayed library, which method comprises incubating a phagemidclone corresponding to the peptide with the polypeptide in the presenceof the ligand, serially diluting the phage, and measuring the degree towhich binding of the phagemid clone to the ligand is inhibited by thepeptide, wherein a phagemid clone that is inhibited only at low phageconcentrations has a higher affinity for the ligand than a phagemidclone that is inhibited at both high and low phage concentrations.

In another embodiment herein, a method for directing endogenous IGFeither away from, or towards, a particular site in a mammal comprisesadministering to the mammal an effective amount of the compound hereinthat is specific for an IGFBP that is either prevalent at, or absentfrom, the site.

A further embodiment is a method for detecting endogenous or exogenousIGF bound to an IGF binding protein or the amount of a compound thatbinds to an IGF binding protein and does not bind to a human IGFreceptor bound to an IGF binding protein or detecting the level ofunbound IGF in a biological fluid comprising:

(a) contacting the fluid with 1) a means for detecting the compoundattached to a solid-phase carrier, wherein the means is specific for thecompound such that in the presence of the compound the IGF binding sitesremain available on the compound for binding to the IGF binding protein,thereby forming a complex between the means and the IGF binding protein;and 2) the compound for a period of time sufficient to saturate allavailable IGF binding sites on the IGF binding protein, thereby forminga saturated complex;

(b) contacting the saturated complex with a detectably labeled secondmeans which is specific for the IGF binding protein when the compound isbound to the IGF binding protein; and

(c) quantitatively analyzing the amount of the labeled means bound as ameasure of the IGFBP in the biological fluid, and therefore as a measureof the amount of bound compound and IGF binding protein, bound IGF andIGF binding protein, or active IGF present in the fluid.

There has been much debate as to the role of the IGFBPs in the action ofan IGF. The activity of the IGFs in various situations has been shown tobe either inhibited, enhanced, or unaffected by the presence of theIGFBPs. Jones and Clemmons, supra; Bach and Rechler, supra. It has beenunclear if the presence of IGFBPs is obligatory for some actions of theIGFs. For some actions it was thought possible that it was necessary forthe IGFs to be bound to the IGFBPs, or that it was necessary for theIGFBPs to be present if IGF-I were to be fully active. Before thepresent studies it was therefore unclear as to what would be the netbiological effect in vivo of administering molecules that prevent theinteraction of an IGF with any one of its IGFBPs.

The compounds herein are superior to IGF mutants such as des(1-3)IGF-I,since the latter have short half-lives and effects, whereas thecompounds herein have longer half lives and effects, and, if they bindto IGFBPs, this binding avoids normal renal filtration which wouldotherwise eliminate short peptides and other small molecules rapidly.Further, administering the compound herein together with exogenous GH orGH secretagogues would have the advantage of minimizing diabetogeniceffects of such GH and secretagogues. Yet another advantage of thecompounds herein is that there is a ceiling of the effects of the IGFagonist compound herein. That is, it cannot exert more effects than themaximum capacity of IGFBPs to carry IGFs, unlike IGF-I, which can haveunwanted side effects if used in large concentrations over its maximumefficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO:17) and translatedamino acid sequence (SEQ ID NO:18) of the lamB signal connected at the5' end of DNA encoding an IGF-I mutant designated herein as Y24L,Y31A,or (Leu²⁴, Ala³¹)hIGF-I, or IGF-M, where the Tyr at position 24 ischanged to Leu and the Tyr at position 31 is changed to Ala.

FIG. 2 depicts the construction of the plasmid pIGFMI from the vectorfragment of p131TGF, from a 120-bp and a 190-bp fragment of pBKIGF-2B,and from a synthetic piece of DNA.

FIGS. 3A-3D depicts the full nucleotide sequence of pIGFMI (SEQ IDNO:19).

FIG. 4 is a standard curve for receptor phosphorylation when IGF-I(squares) or (Leu²⁴, Ala³¹)hIGF-I (circles) is added to MCF-7 cells in aKIRA phosphorylation assay.

FIG. 5 shows thymidine incorporation into mouse 3T3 cells using IGF-I or(Leu²⁴, Ala³¹)hIGF-I.

FIG. 6 shows the binding affinity of (Leu²⁴, Ala³¹)hIGF-I to IGFBP-1.

FIG. 7 shows the binding affinity of (Leu²⁴, Ala³¹)hIGF-I to IGFBP-3.

FIGS. 8A and 8B depict the responses in plasma glucose (FIG. 8A) andplasma insulin (FIG. 8B) of normal rats treated, in a first study, with(Leu²⁴, Ala³¹)hIGF-I (solid circles) or a control (PBS) (open squares),expressed as a percentage of the values in the pre-treatment bloodsamples which were averaged and set at 100%.

FIGS. 9A and 9B depict the responses in plasma glucose (FIG. 9A) andplasma insulin (FIG. 9B) of normal rats treated, in a second study, with(Leu²⁴, Ala³¹)hIGF-I (solid triangles), or IGF-I (solid circles), or acontrol (open squares), expressed as a percentage of the values in thepre-treatment blood samples which were averaged and set at 100%.

FIGS. 10A and 10B show the percentage change in plasma insulin frombaseline (set as 100%) (FIG. 10A) and the percentage change in plasmaglucose from baseline (set as 100%) (FIG. 10B) of diabetic rats treatedwith (Leu²⁴, Ala³¹)hIGF-I, IGF-I, or a control.

FIGS. 11A and 11B show the percentage change in plasma insulin frombaseline (set as 100%) (FIG. 11A) and the percentage change in plasmaglucose from baseline (set as 100%) (FIG. 11B) of diabetic (ZDF) ratstreated, in a second study, with (Leu²⁴, Ala³¹)hIGF-I, IGF-I, or acontrol.

FIG. 12 shows the final body weights of rats treated with (Leu²⁴Ala³¹)hIGF-I at two doses, with GH, with a combination of GH and (Leu²⁴,Ala³¹)hIGF-I, or with a control over seven days.

FIGS. 13A and 13B show the weight of the spleen in the study describedabove for FIG. 12, with FIG. 13A showing absolute spleen weight and FIG.13B showing spleen weight expressed as a percentage of body weight.

FIGS. 14A and 14B show the weight of the thymus in the study describedabove for FIG. 12, with FIG. 14A showing absolute thymus weight and FIG.14B showing thymus weight expressed as a percentage of body weight.

FIGS. 15A and 15B show the weight of the heart in the study describedabove for FIG. 12, with FIG. 15A showing absolute heart weight and FIG.15B showing heart weight expressed as a percentage of body weight.

FIG. 16 shows the epiphyseal plate widths from the five treatment groupsin the study described for FIG. 12.

FIGS. 17A and 17B show the amount of rat IGF-I (FIG. 17A) and the amountof total IGF-I (FIG. 17B) in the blood in the five treatment groups inthe study described for FIG. 12.

FIG. 18 depicts the final body weights over seven days of dwarf ratstreated with placebo (open squares), (Leu²⁴, Ala³¹)hIGF-I (opentriangles), IGF-I (open circles), a combination of (Leu²⁴, Ala³¹)hIGF-Iand IGF-I (solid circles), GH (half open/half solid squares), and acombination of (Leu²⁴, Ala³¹)hIGF-I and GH (solid squares).

FIGS. 19A and 19B show the weight of the spleen in the study describedabove for FIG. 18, with FIG. 19A showing absolute spleen weight and FIG.19B showing spleen weight expressed as a percentage of body weight.

FIGS. 20A and 20B show the weight of the kidney in the study describedabove for FIG. 18, with FIG. 20A showing absolute kidney weight and FIG.20B showing kidney weight expressed as a percentage of body weight.

FIGS. 21A and 21B show the amount of rat IGF-I (FIG. 21A) and the amountof total IGF-I (FIG. 21B) in the blood in the six treatment groups inthe study described for FIG. 18.

FIGS. 22A and 22B depict body weight gain (FIG. 22A) and percentincrease in blood glucose levels (FIG. 22B) over a longer time period ofdiabetic rats treated with excipient control (solid triangles), with(Leu²⁴, Ala³¹)hIGF-I at 150 μg, tid (three times daily) (open squares),with (Leu²⁴, Ala³¹)hIGF-I at 50 μg, tid (open circles), and with IGF-Iat 150 μg, tid (solid circles).

FIGS. 23A and 23B show the increase in blood glucose levels (FIG. 23A)and the changes in insulin levels (FIG. 23B) in the study described forFIG. 22. The control is indicated by black bars, the mutant at 150 μg bydark stippled bars, the mutant at 50 μg by light stippled bars, and theIGF-I by open bars.

FIGS. 24A-C depicts the partial DNA sequence (SEQ ID NO: 20) of plasmidpt4.g8 used as a template to construct a phage library. Also shown isthe amino acid sequence (SEQ ID NO:21) of an antibody-recognizable(gD-tag) peptide fused to g8p of bacteriophage M13.

FIG. 25 shows gene-8 naive phage library enrichments with a selectionusing four library pools each and the targets IGF-I, IGFBP-1, andIGFBP-3.

FIG. 26 shows an IGF-I blocking assay using g8-phage peptides fromIGFBP-3 selections, where the phage titration is with 100 nM IGF-I. Inthe Figure, the open circles are peptide 4A3.1, the open triangles arepeptide 4B3.4, the open squares are peptide 4C3.2, the solid circles arepeptide 4D3.3, the solid triangles are peptide 4D3.4, and the solidsquares are peptide 4D3.5.

FIG. 27 shows an IGF-I blocking assay using g8-phage peptides fromIGFBP-3 selections, where the phage titration is without IGF-I. Thedesignations for the peptides are the same as those described above forFIG. 26.

FIG. 28 shows an IGF-I blocking assay using a g8-phage peptide from anIGFBP-1 selection (peptide bp1-01), where the phage titration is with 1μM IGF-I.

FIG. 29 shows an IGF-I blocking assay using g8-phage peptides fromIGFBP-3 selections, where the peptides (4C3.2, 4D3.8, 4D3.9, 4D3.11, and4D3.12) are from a NEUTRAVIDIN™/DTT selection. The solid bars are with100 μM IGF-I and the open bars are without IGF-I.

FIG. 30 shows an IGF-I blocking assay using g8-phage peptides fromIGFBP-3 selections where the peptides (indicated on the x axis) are fromdirect-coat/HCl selection. The solid bars are with 100 μM IGF-I and theopen bars are without IGF-I.

FIG. 31 depicts a competition assay of IGFBP-3 inhibition by a peptidebinding to IGFBP-3 (designated BP3-01) using a BIAcore™surface-plasmon-resonance device to measure free binding protein. Thecircles indicate 800 response units (RU) of IGF-I and the squaresindicate 400 RU of immobilized IGF-I.

FIG. 32 depicts a competition assay of IGFBP-3 inhibition by a peptidebinding to IGFBP-3 (designated BP3-02) using a BIAcore™surface-plasmon-resonance device to measure free binding protein. Thecircles indicate 800 RU of IGF-I and the squares indicate 400 RU ofimmobilized IGF-I.

FIG. 33 shows inhibition of biotinylated IGFBP-1 binding to IGF-I onplates by three peptides that bind to IGFBP-1 or IGFBP-3 but do not bindto the Type 1 IGF receptor (bp1-01: solid circles, bp1-02: open circles,and bp3-01-ox: open triangles).

FIG. 34 shows inhibition of biotinylated IGFBP-3 binding to IGF-I onplates by two peptides that bind to IGFBP-1 but not to the Type 1 IGFreceptor (bp1-01 (bp3): solid circles, and bp1-02 (bp3): open circles).

FIG. 35 shows a radiolabeled IGF-I plate assay of the ability of twopeptides that bind to IGFBP-3 but not to the Type I IGF receptor(bp3-01-ox: circles, and bp3-02-ox: squares) to inhibit IGFBP-3.

FIG. 36 shows a radiolabeled IGF-I plate assay of the ability of the twoIGFBP-3 binding peptides described for FIG. 35 to inhibit IGFBP-1(symbols are the same).

FIG. 37 depicts KIRA assays of IGF-I activity using three peptides(bp1-01: squares, bp1-02: circles, and bp03-ox:

triangles). FIG. 37A depicts the peptides alone, FIG. 37B depicts thepeptides plus IGF-I plus IGFBP-1, FIG. 37C depicts the peptides plusIGF-I, and FIG. 37D depicts the peptides plus IGF-I plus IGFBP-3.

FIG. 38 depicts an IGF-II competition assay of IGFBP-3 inhibition byfour peptides, designated bp3-01-ox (open squares), BP14 (open circles),BP15 (closed circles), and BP17 (closed squares), using a BIAcore™surface-plasmon-resonance device to measure free binding protein. Eachpeptide was tested using 20 nM IGFBP-3 and approximately 1500 RU ofimmobilized IGF-II.

FIG. 39 depicts a one-dimensional ¹ H NMR spectrum of a peptide bindingto IGFBP-1 (bp1-01) in H₂ O solution at 30° C.

FIGS. 40A and 40B disclose a three-dimensional model of the structure ofthe peptide bp1-01 in solution. These are stereoviews of arepresentative structure of bp1-01 from the ensemble of structurescalculated using restraints derived from NMR data. The backbone fold isdepicted as a ribbon, and all side-chain heavy atoms are shown; eachnon-glycine residue is labeled. The two views differ by approximately90°. The relatively flat hydrophobic surface (on the left in FIG. 40A,and towards the viewer in FIG. 40B) is involved in self association, andmay also be involved in IGFBP-1 binding.

FIG. 41 is a chromatogram demonstrating the ability of the mutant(Leu²⁴, Ala³¹)hIGF-I to displace ¹²⁵ I-IGF-I from endogenous IGFBPspresent in serum from normal humans. Data are expressed as cpm perfraction (n=1). In the figure, the solid lines with circles are themutant at time 0 of incubation, the long dashed lines with circles arethe mutant at time 0.5 hr of incubation, the shorter dashed lines withdiamonds are the mutant at time 3 hr of incubation, and the shortestdotted lines with squares are the mutant at time 16 hr of incubation.

FIG. 42 shows the concentrations of IGF-I in the blood of type IIdiabetic patients treated with placebo (p) or 10 (1), 20 (2), 40 (4), or80 (8) μg/kg of rhIGF-I for 12 weeks by twice daily subcutaneousinjection.

FIG. 43 shows the concentrations of IGF-II in the blood of the patientstreated as described for FIG. 42.

FIG. 44 shows the concentrations of IGFBP-3 in the blood of the patientstreated as described for FIG. 42.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. Definitions

As used herein, "mammal" for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic, and farm animals,and zoo, sports, or pet animals, such as dogs, horses, cats, sheep,pigs, cows, etc. The preferred mammal herein is a human. The term"non-adult" refers to mammals that are from perinatal age (such aslow-birth-weight infants) up to the age of puberty, the latter beingthose that have not yet reached full growth potential.

As used herein, "IGF" refers to native insulin-like growth factor-I andnative insulin-like growth factor-II as well as natural variants thereofsuch as brain IGF, otherwise known as des(1-3)IGF-I.

As used herein, "IGF-I" refers to insulin-like growth factor-I from anyspecies, including bovine, ovine, porcine, equine, and human, preferablyhuman, and, if referring to exogenous administration, from any source,whether natural, synthetic, or recombinant. Human native-sequence,mature IGF-I, more preferably without a N-terminal methionine, isprepared, e.g., by the process described in EP 230,869 published Aug. 5,1987; EP 128,733 published Dec. 19, 1984; or EP 288,451 published Oct.26, 1988. More preferably, this native-sequence IGF-I is recombinantlyproduced and is available from Genentech, Inc., South San Francisco,Calif. for clinical investigations.

As used herein, "IGF-II" refers to insulin-like growth factor-II fromany species, including bovine, ovine, porcine, equine, and human,preferably human, and, if referring to exogenous administration, fromany source, whether natural, synthetic, or recombinant. It may beprepared by the method described in, e.g., EP 128,733, supra.

An "IGFBP" or an "IGF binding protein" refers to a protein orpolypeptide normally associated with or bound or complexed to IGF-I orIGF-II, whether or not it is circulatory (i.e., in serum or tissue).Such binding proteins do not include receptors. This definition includesIGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5, IGFBP-6, Mac 25 (IGFBP-7),and prostacyclin-stimulating factor (PSF) or endothelial cell-specificmolecule (ESM-1), as well as other proteins with high homology toIGFBPs. Mac 25 is described, for example, in Swisshelm et al., Proc.Natl. Acad. Sci. USA, 92: 4472-4476 (1995) and Oh et al., J. Biol.Chem., 271: 30322-30325 (1996). PSF is described in Yamauchi et al.,Biochemical Journal, 303: 591-598 (1994). ESM-1 is described in Lassalleet al., J. Biol. Chem., 271: 20458-20464 (1996). For other identifiedIGFBPs, see, e.g., EP 375,438 published Jun. 27, 1990; EP 369,943published May 23, 1990; WO 89/09268 published Oct. 5, 1989; Wood et al.,Molecular Endocrinology, 2: 1176-1185 (1988); Brinkman et al., The EMBOJ., 7: 2417-2423 (1988); Lee et al., Mol. Endocrinol., 2: 404-411(1988); Brewer et al., BBRC, 152: 1289-1297 (1988); EP 294,021 publishedDec. 7, 1988; Baxter et al., BBRC, 147: 408-415 (1987); Leung et al.,Nature, 330: 537-543 (1987); Martin et al., J. Biol. Chem., 261:8754-8760 (1986); Baxter et al., Comp. Biochem. Physiol., 91B: 229-235(1988); WO 89/08667 published Sep. 21, 1989; WO 89/09792 published Oct.19, 1989; and Binkert et al., EMBO J., 8: 2497-2502 (1989).

The term "body fluid" refers to a biological sample of liquid from amammal, preferably from a human. Such fluids include aqueous fluids suchas serum, plasma, lymph fluid, synovial fluid, follicular fluid, seminalfluid, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid,saliva, sputum, tears, perspiration, mucus, tissue culture medium,tissue extracts, and cellular extracts.

As used herein, "human IGF receptor" refers to any receptor for an IGFfound in humans and includes the Type 1 and Type 2 IGF receptors inhumans to which both human IGF-I and IGF-II bind, such as the placentalType 1 IGF-I receptor, etc.

A compound that "prevents the interaction of an IGF with any one of itsIGFBPs" refers to a molecule that increases serum and tissue levels ofbiologically active IGF, no matter how this increase occurs. Forinstance, the compound may partially or completely displace active IGFfrom a complex in which the IGF is bound to one or more of its IGFBPs.The compound under this definition may bind to an IGFBP, and possiblythereby act to displace an endogenous IGF formerly bound to the IGFBP,or it may bind to an IGF itself at a site remote from that involved inreceptor interactions so as to inhibit or prevent the interaction of theIGF with one or more of its IGFBPs, but not inhibit or prevent theinteraction of the IGF with any of its receptors. Further, while thecompound will occupy the IGFBPs, the effect on the ternary complex willdepend on whether the binary complexes can form ternary ones. IGFagonist compounds that can form complexes with ALS will replace IGFs butnot affect the concentration of IGFBP-3 or of ternary complexes. IGFagonist compounds that cannot form complexes with ALS will occupyIGFBP-3, and the amount of ALS/IGFBP-3/IGF complex will be reduced. Thismay differ between full-length IGF mutants which may form ternarycomplexes and small peptides, which might not. With respect to thestructure of one exemplary peptide herein and its interaction with anIGFBP, see Example 9 below.

A compound that "binds to IGF binding protein" refers to a compound thatbinds an IGFBP to at least some degree, whether with high affinity ornot.

A compound that "does not bind to a human IGF receptor" does not bind atall to any such receptor, or binds to such receptor with an affinitymore than about 200-fold less than wild-type human IGF-I (hIGF-I) orwild-type human IGF-II (hIGF-II) binds to such receptor. Preferably, thecompound binds to such receptor with an affinity of more than about250-fold less than wild-type hIGF-I or hIGF-II binds to the samereceptor or does not bind at all. Such a compound is additionallydefined as one that does not phosphorylate the human Type 1 IGF receptorand does not stimulate the mouse IGF-I receptor as measured by thymidineuptake into mouse 3T3 cells using the KIRA and mouse 3T3 assays ofExample 1, i.e, the compound acts like (Leu²⁴,Ala³¹)hIGF-I or binds thereceptor even less than this mutant in these assays. Further, IGF-IIcould be construed as an IGF-I agonist, and IGF-I could be construed asan IGF-II agonist. However, both IGF-I and IGF-II bind to the Type 1 IGFreceptor, and thus are both receptor-active molecules and not within thescope of the compounds as defined herein.

A "disorder" is any condition that would benefit from treatment with anIGF, including but not limited to, for example, lung diseases,hyperglycemic disorders as set forth below, renal disorders, such asacute and chronic renal insufficiency, end-stage chronic renal failure,glomerulonephritis, interstitial nephritis, pyelonephritis,glomerulosclerosis, e.g., Kimmelstiel-Wilson in diabetic patients andkidney failure after kidney transplantation, obesity, GH-insufficiency,Turner's syndrome, Laron's syndrome, short stature, undesirable symptomsassociated with aging such as obesity and increased fat mass-to-leanratios, immunological disorders such as immunodeficiencies includingdecreased CD4 counts and decreased immune tolerance orchemotherapy-induced tissue damage, bone marrow transplantation,diseases or insufficiencies of cardiac structure or function such asheart disfunctions and congestive heart failure, neuronal, neurological,or neuromuscular disorders, e.g., peripheral neuropathy, multiplesclerosis, muscular dystrophy, or myotonic dystrophy, and catabolicstates associated with wasting caused by any condition, including, e.g.,trauma or wounding or infection such as with a bacterium or human virussuch as HIV, wounds, skin disorders, gut structure and function thatneed restoration, and so forth. The disorder being treated may be acombination of two or more of the above disorders. The preferreddisorders targeted for treatment herein are diabetes and obesity, heartdisfunctions, kidney disorders, neurological disorders, whole bodygrowth disorders, and immunological disorders.

As used herein, the term "hyperglycemic disorders" refers to all formsof diabetes and disorders resulting from insulin resistance, such asType I and Type II diabetes, as well as severe insulin resistance,hyperinsulinemia, and hyperlipidemia, e.g., obese subjects, andinsulin-resistant diabetes, such as Mendenhall's Syndrome, WernerSyndrome, leprechaunism, lipoatrophic diabetes, and other lipoatrophies.The preferred hyperglycemic disorder is diabetes, especially Type I andType II diabetes. "Diabetes" itself refers to a progressive disease ofcarbohydrate metabolism involving inadequate production or utilizationof insulin and is characterized by hyperglycemia and glycosuria.

As used herein, the term "treating" refers to both therapeutic treatmentand prophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those prone to havingthe disorder or diagnosed with the disorder or those in which thedisorder is to be prevented. Consecutive treatment or administrationrefers to treatment on at least a daily basis without interruption intreatment by one or more days. Intermittent treatment or administration,or treatment or administration in an intermittent fashion, refers totreatment that is not consecutive, but rather cyclic in nature. Thetreatment regime herein can be either consecutive or intermittent.

As used herein, the term "hypoglycemic agent" refers to compounds thatare useful for regulating glucose metabolism, preferably oral agents.More preferred herein for human use are insulin and the sulfonylureaclass of oral hypoglycemic agents, which cause the secretion of insulinby the pancreas. Examples include glyburide, glipizide, and gliclazide.In addition, agents that enhance insulin sensitivity or are insulinsensitizing, such as biguanides (including metformin and phenformin) andthiazolidenediones such as REZULIN™ (troglitazone) brandinsulin-sensitizing agent, and other compounds that bind to thePPARgamma nuclear receptor, are within this definition, and also arepreferred.

As used herein, "insulin" refers to any form of insulin from anyspecies, and whether natively or synthetically or recombinantly derived.Preferably it is NPH insulin.

As used herein, "active" or "biologically active" IGF in the context ofchanging serum and tissue levels of endogenous IGF refers to IGF thatbinds to its receptor or otherwise causes a biological activity tooccur, such as those biological activities of endogenous or exogenousIGF referred to above.

A "molecule" or "compound" that prevents interaction of an IGF with anyone of its IGFBPs and does not bind a human IGF receptor includesmolecules with high oral bioavailability, exemplified by GHsecretagogues, organic chemical molecules modeled after the3-dimensional model given herein, and peptides as defined below. Suchcompounds are also referred to herein as "IGF agonists" or "IGF agonistcompounds".

"Peptides" include molecules having at least two amino acids and includepolypeptides having at least about 50 amino acids. Preferably, thepeptides have about 10 to about 25 amino acids, more preferably about12-25, and most preferably about 15-25 amino acids. The definitionincludes peptide derivatives, their salts, or optical isomers.

"Growth hormone releasing peptides or factors" ("GHRP" or "GHRF") aredescribed below, as are secretagogues. A "growth hormone releasinghormone" ("GHRH") can be any hormone that releases GH from the cells ortissue. "Growth hormone in combination with a growth hormone bindingprotein" ("GH" plus "GHBP") means a GH complexed with or otherwiseassociated with one of its binding proteins. Similarly, "IGF incombination with an IGF binding protein" ("IGF" plus "IGFBP") refers toan IGF complexed with or otherwise associated with one of its IGFBPs.

B. Modes for Carrying Out the Invention

The invention herein relates to a compound or molecule that preventsinteraction of an IGF with one or more of its IGFBPs and does not bindto a human IGF receptor, excluding (1-27,gly⁴,38-70)-hIGF-I, excludingantibodies against (that bind to) an IGFBP, excluding antibodies against(that bind to) an IGF, and excluding peptides having the native sequenceof human IGF-I with the tyrosine residues at positions 24, 31, and/or 60replaced or deleted. Preferably, the compound binds to an IGFBP and/orto an IGF, especially to IGFBP-1, or to IGFBP-3, or to both IGFBP-1 andIGFBP-2, or to IGF-I, or to IGF-II, or to both IGF-I and IGF-II, or toIGFBP-1 or -3 and IGF-I or -II. Also preferably, the compound is apeptide. More preferably, it is a peptide that binds to an IGFBP, morepreferably to a serum IGFBP. Also, preferably, the compound reducesplasma insulin secretion, reduces plasma GH, or reduces blood glucoselevels in a mammal. More preferably, the peptide has about 10 to about25 amino acid residues and/or has a cysteine residue at position 5, 6,7, or 8 numbered from its N-terminus or a cysteine residue at position5, 6, 7, or 8 numbered from its C-terminus, or both such cysteineresidues, or a cysteine residue at position 2 numbered from itsN-terminus. Preferably, the peptide described above has about 12 to 25,more preferably 15 to 25, amino acid residues. More preferably, thepeptide has a tryptophan residue at position 1, 2, 3, 4, or 5 numberedfrom its N-terminus. Still more preferably, the peptide additionally hasa valine, serine, or glutamine residue N-terminal to the N-terminalcysteine residue. Even more preferably, the peptide is such that theresidue N-terminal to the N-terminal cysteine residue is a valineresidue. Still more preferably, the peptide has a glycine-proline orvaline-alanine sequence of residues beginning at a position that isthree residues C-terminal to the N-terminal cysteine. This peptidefurther preferably has a tryptophan residue within three residuesC-terminal to the glycine-proline or valine-alanine sequence ofresidues. This peptide more preferably has a leucine or valine residuewithin two residues N-terminal to the C-terminal cysteine residue. Thispeptide even more preferably has a tryptophan residue at position 2 or 3numbered from its C-terminus.

Also preferred is a peptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOS:1-16. Preferably, this peptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.More preferably, this peptide comprises an amino acid sequence that isSEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:15, or SEQ ID NO:16. Still more preferably, this peptide comprisesan amino acid sequence that is SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:15,or SEQ ID NO:16. Most preferably, this peptide comprises an amino acidsequence that is SEQ ID NO:15 or SEQ ID NO:16.

The compound preferably excludes any IGF-I analogs of the formula:BC^(n), A

wherein B is the B domain of IGF-I or a functional analog thereof, C isthe C domain of IGF-I or a functional analog thereof, n is the number ofamino acids in the C domain and is from about 6 to about 12, and A isthe A domain of IGF-I or a functional analog thereof. The compoundsherein specifically exclude (Leu²⁴)IGF-I and (Ser²⁴)IGF-I. The compoundsalso exclude a B-chain human IGF-I mutant in which the first 16 aminoacids of IGF-I are replaced with the first 17 amino acids of the B-chainof insulin; (Gln³,Ala⁴)IGF-I; (Tyr¹⁵,Leu¹⁶)IGF-I;(Gln³,Ala⁴,Tyr¹⁵,Leu¹⁶)IGF-I; (1-62)IGF-I, which lacks thecarboxyl-terminal 8-amino-acid D region of IGF-I;(1-27,Gly⁴,38-62)IGF-I, with a C region glycine replacement and a Dregion deletion; (Ile⁴¹,Glu⁴⁵,Gln⁴⁶, Thr⁴⁹,Ser⁵⁰, Ile⁵¹, Ser⁵³, Tyr⁵⁵,Gln⁵⁶)IGF-I, an A chain mutant in which residue 41 is changed fromthreonine to isoleucine and residues 42-56 of the A region are replaced;(Thr⁴⁹,Ser⁵⁰,Ile⁵¹)IGF-I; and (Tyr⁵⁵,Gln⁵⁶)IGF-I.

The compounds herein that are not peptides may be made by chemicalsynthesis or other appropriate methods in the art for makingnon-peptidyl molecules with high oral bioavailability (such as GHsecretagogues) and other orally active organic molecules.

The peptides of this invention can be made by chemical synthesis or byemploying recombinant technology. These methods are known in the art.Chemical synthesis, especially solid phase synthesis, is preferred forshort (e. g., less than 50 residues) peptides or those containingunnatural or unusual amino acids such as D-Tyr, Ornithine, amino adipicacid, and the like. Recombinant procedures are preferred for longerpolypeptides. When recombinant procedures are selected, a synthetic genemay be constructed de novo or a natural gene may be mutated by, forexample, cassette mutagenesis. Set forth below are exemplary generalrecombinant procedures.

From a purified IGF and its amino acid sequence, for example, an IGFagonist that is a peptidyl mutant of an IGF may be produced usingrecombinant DNA techniques. These techniques contemplate, in simplifiedform, taking the gene, either natural or synthetic, encoding thepeptide; inserting it into an appropriate vector; inserting the vectorinto an appropriate host cell; culturing the host cell to causeexpression of the gene; and recovering or isolating the peptide producedthereby. Preferably, the recovered peptide is then purified to asuitable degree.

Somewhat more particularly, the DNA sequence encoding a peptidyl IGFagonist is cloned and manipulated so that it may be expressed in aconvenient host. DNA encoding parent polypeptides can be obtained from agenomic library, from cDNA derived from mRNA from cells expressing thepeptide, or by synthetically constructing the DNA sequence (Sambrook etal., Molecular Cloning: A Laboratory Manual (2d ed.), Cold Spring HarborLaboratory, N.Y., 1989).

The parent DNA is then inserted into an appropriate plasmid or vectorwhich is used to transform a host cell. In general, plasmid vectorscontaining replication and control sequences which are derived fromspecies compatible with the host cell are used in connection with thosehosts. The vector ordinarily carries a replication site, as well assequences which encode proteins or peptides that are capable ofproviding phenotypic selection in transformed cells.

For example, E. coli may be transformed using pBR322, a plasmid derivedfrom an E. coli species. Mandel et al., J. Mol. Biol. 53: 154 (1970).Plasmid pBR322 contains genes for ampicillin and tetracyclineresistance, and thus provides easy means for selection. Other vectorsinclude different features such as different promoters, which are oftenimportant in expression. For example, plasmids pKK223-3, pDR720, andpPL-lambda represent expression vectors with the tac, trp, or PLpromoters that are currently available (Pharmacia Biotechnology).

A preferred vector is pB0475. This vector contains origins ofreplication for phage and E. coli that allow it to be shuttled betweensuch hosts, thereby facilitating both mutagenesis and expression.Cunningham et al., Science, 243: 1330-1336 (1989); U.S. Pat. No.5,580,723. Other preferred vectors are pRIT5 and PR1T2T (PharmaciaBiotechnology). These vectors contain appropriate promoters followed bythe Z domain of protein A, allowing genes inserted into the vectors tobe expressed as fusion proteins.

Other preferred vectors can be constructed using standard techniques bycombining the relevant traits of the vectors described above. Relevanttraits include the promoter, the ribosome binding site, the decorsin orornatin gene or gene fusion (the Z domain of protein A and decorsin orornatin and its linker), the antibiotic resistance markers, and theappropriate origins of replication.

The host cell may be prokaryotic or eukaryotic. Prokaryotes arepreferred for cloning and expressing DNA sequences to produce parentIGF-I polypeptide, segment-substituted peptides, residue-substitutedpeptides, and peptide variants. For example, E. coli K12 strain 294(ATCC No. 31446) may be used as well as E. coli B, E. coli X1776 (ATCCNo. 31537), and E. coli c600 and c600hfl, E. coli W3110 (F-, gamma-,prototrophic/ATCC No. 27325), bacilli such as Bacillus subtilis, andother enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species. The preferred prokaryote isE. coli W3110 (ATCC 27325). When expressed by prokaryotes the peptidestypically contain an N-terminal methionine or a formyl methionine andare not glycosylated. In the case of fusion proteins, the N-terminalmethionine or formyl methionine resides on the amino terminus of thefusion protein or the signal sequence of the fusion protein. Theseexamples are, of course, intended to be illustrative rather thanlimiting.

In addition to prokaryotes, eukaryotic organisms, such as yeastcultures, or cells derived from multicellular organisms may be used. Inprinciple, any such cell culture is workable. However, interest has beengreatest in vertebrate cells, and propagation of vertebrate cells inculture (tissue culture) has become a reproducible procedure. TissueCulture, Academic Press, Kruse and Patterson, editors (1973). Examplesof such useful host cell lines are VERO and HeLa cells, Chinese HamsterOvary (CHO) cell lines, W138, 293, BHK, COS-7, and MDCK cell lines.

A variation on the above procedures contemplates the use of genefusions, wherein the gene encoding the desired peptide is associated, inthe vector, with a gene encoding another protein or a fragment ofanother protein. This results in the desired peptide being produced bythe host cell as a fusion with another protein or peptide. The "other"protein or peptide is often a protein or peptide which can be secretedby the cell, making it possible to isolate and purify the desiredpeptide from the culture medium and eliminating the necessity ofdestroying the host cells which arises when the desired peptide remainsinside the cell. Alternatively, the fusion protein can be expressedintracellularly. It is useful to use fusion proteins that are highlyexpressed.

The use of gene fusions, though not essential, can facilitate theexpression of heterologous peptides in E. coli as well as the subsequentpurification of those gene products. Harris, in Genetic Engineering,Williamson, R., Ed. (Academic Press, London, Vol. 4, 1983), p. 127;Ljungquist et al., Eur. J. Biochem., 186: 557-561 (1989) and Ljungquistet al., Eur. J. Biochem., 186: 563-569 (1989). Protein A fusions areoften used because the binding of protein A, or more specifically the Zdomain of protein A, to IgG provides an "affinity handle" for thepurification of the fused protein. It has also been shown that manyheterologous proteins are degraded when expressed directly in E. coli,but are stable when expressed as fusion proteins. Marston, Biochem J.,240: 1 (1986).

Fusion proteins can be cleaved using chemicals, such as cyanogenbromide, which cleaves at a methionine, or hydroxylamine, which cleavesbetween an Asn and Gly residue. Using standard recombinant DNAmethodology, the nucleotide base pairs encoding these amino acids may beinserted just prior to the 5' end of the gene encoding the desiredpeptide.

Alternatively, one can employ proteolytic cleavage of fusion protein.Carter, in Protein Purification: From Molecular Mechanisms toLarge-Scale Processes, Ladisch et al., eds. (American Chemical SocietySymposium Series No. 427, 1990), Ch 13, pages 181-193.

Proteases such as Factor Xa, thrombin, and subtilisin or its mutants,and a number of others have been successfully used to cleave fusionproteins. Typically, a peptide linker that is amenable to cleavage bythe protease used is inserted between the "other" protein (e.g., the Zdomain of protein A) and the desired peptide. Using recombinant DNAmethodology, the nucleotide base pairs encoding the linker are insertedbetween the genes or gene fragments coding for the other proteins.Proteolytic cleavage of the partially purified fusion protein containingthe correct linker can then be carried out on either the native fusionprotein, or the reduced or denatured fusion protein.

The peptide may or may not be properly folded when expressed as a fusionprotein. Also, the specific peptide linker containing the cleavage sitemay or may not be accessible to the protease. These factors determinewhether the fusion protein must be denatured and refolded, and if so,whether these procedures are employed before or after cleavage.

When denaturing and refolding are needed, typically the peptide istreated with a chaotrope, such a guanidine HCl, and is then treated witha redox buffer, containing, for example, reduced and oxidizeddithiothreitol or glutathione at the appropriate ratios, pH, andtemperature, such that the peptide is refolded to its native structure.

When peptides are not prepared using recombinant DNA technology, theyare preferably prepared using solid-phase synthesis, such as thatgenerally described by Merrifield, J. Am. Chem. Soc., 85: 2149 (1963),although other equivalent chemical syntheses known in the art areemployable. Solid-phase synthesis is initiated from the C-terminus ofthe peptide by coupling a protected α-amino acid to a suitable resin.Such a starting material can be prepared by attaching anα-amino-protected amino acid by an ester linkage to a chloromethylatedresin or a hydroxymethyl resin, or by an amide bond to a BHA resin orMBHA resin. The preparation of the hydroxymethyl resin is described byBodansky et al., Chem. Ind. (London), 38: 1597-1598 (1966).Chloromethylated resins are commercially available from BioRadLaboratories, Richmond, Calif. and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart et al., "Solid PhasePeptide Synthesis" (Freeman & Co., San Francisco 1969), Chapter 1, pp.1-6. BHA and MBHA resin supports are commercially available and aregenerally used only when the desired polypeptide being synthesized hasan unsubstituted amide at the C-terminus.

The amino acids are coupled to the peptide chain using techniques wellknown in the art for the formation of peptide bonds. One method involvesconverting the amino acid to a derivative that will render the carboxylgroup more susceptible to reaction with the free N-terminal amino groupof the peptide fragment. For example, the amino acid can be converted toa mixed anhydride by reaction of a protected amino acid withethylchloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, pivaloyl chloride or like acid chlorides.Alternatively, the amino acid can be converted to an active ester suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole.

Another coupling method involves use of a suitable coupling agent suchas N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide. Otherappropriate coupling agents, apparent to those skilled in the art, aredisclosed in E. Gross & J. Meienhofer, The Peptides: Analysis,Structure, Biology, Vol. I: Major Methods of Peptide Bond Formation(Academic Press, New York, 1979).

It should be recognized that the α-amino group of each amino acidemployed in the peptide synthesis must be protected during the couplingreaction to prevent side reactions involving their active α-aminofunction. It should also be recognized that certain amino acids containreactive side-chain functional groups (e.g., sulfhydryl, amino,carboxyl, and hydroxyl) and that such functional groups must also beprotected with suitable protecting groups to prevent a chemical reactionfrom occurring at that site during both the initial and subsequentcoupling steps. Suitable protecting groups, known in the art, aredescribed in Gross and Meienhofer, The Peptides: Analysis, Structure,Biology, Vol.3: "Protection of Functional Groups in Peptide Synthesis"(Academic Press, New York, 1981).

In the selection of a particular side-chain protecting group to be usedin synthesizing the peptides, the following general rules are followed.An α-amino protecting group (a) must render the α-amino function inertunder the conditions employed in the coupling reaction, (b) must bereadily removable after the coupling reaction under conditions that willnot remove side-chain protecting groups and will not alter the structureof the peptide fragment, and (c) must eliminate the possibility ofracemization upon activation immediately prior to coupling. A side-chainprotecting group (a) must render the side chain functional group inertunder the conditions employed in the coupling reaction, (b) must bestable under the conditions employed in removing the α-amino protectinggroup, and (c) must be readily removable upon completion of the desiredamino acid peptide under reaction conditions that will not alter thestructure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivitywith the agents employed for their removal. For example, certainprotecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl. adamantyloxycarbonyl, andp-methoxybenzyloxycarbonyl are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride inacetic acid, for their removal. Still other protecting groups, such asbenzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require stronger acids,such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetatein trifluoroacetic acid, for their removal. Among the classes of usefulamino acid protecting groups are included:

(1) for an α-amino group, (a) aromatic urethane-type protecting groups,such as fluorenylmethyloxycarbonyl (FMOC) CBZ, and substituted CBZ, suchas, e.g., p-chlorobenzyloxycarbonyl, p-6-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and the like; (b) aliphatic urethane-typeprotecting groups, such as BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenylyl)-isopropyloxycarbonyl, allyloxycarbonyl, and the like;(c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl;and d) allyloxycarbonyl. The preferred α-amino protecting groups are BOCor FMOC.

(2) for the side chain amino group present in Lys, protection may be byany of the groups mentioned above in (1) such as BOC,p-chlorobenzyloxycarbonyl, etc.

(3) for the guanidino group of Arg, protection may be by nitro, tosyl,CBZ, adamantyloxycarbonyl, 2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC.

(4) for the hydroxyl group of Ser, Thr, or Tyr, protection may be, forexample, by C1-C4 alkyl, such as t-butyl; benzyl (BZL); or substitutedBZL, such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl,o-chlorobenzyl, and 2,6-dichlorobenzyl.

(5) for the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using groups such as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

(6) for the imidazole nitrogen of His, the tosyl moiety is suitablyemployed.

(7) for the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl,or 2,6-dichiorobenzyl is suitably employed. The preferred protectinggroup is 2,6-dichlorobenzyl.

(8) for the side chain amino group of Asn or Gln, xanthyl (Xan) ispreferably employed.

(9) for Met, the amino acid is preferably left unprotected.

(10) for the thio group of Cys, p-methoxybenzyl is typically employed.

The C-terminal amino acid, e.g., Lys, is protected at the N-aminoposition by an appropriately selected protecting group, in the case ofLys, BOC. The BOC-Lys-OH can be first coupled to the benzyhydrylamine orchloromethylated resin according to the procedure set forth in Horiki etal., Chemistry Letters, 165-168 1978) or using isopropylcarbodiimide atabout 25° C. for 2 hours with stirring. Following the coupling of theBOC-protected amino acid to the resin support, the α-amino protectinggroup is removed, as by using trifluoroacetic acid (TFA) in methylenechloride or TFA alone. The deprotection is carried out at a temperaturebetween about 0° C. and room temperature. Other standard cleavingreagents, such as HCl in dioxane, and conditions for removal of specificα-amino protecting groups are described in the literature.

After removal of the α-amino protecting group, the remaining α-amino andside-chain protected amino acids are coupled stepwise within the desiredorder. As an alternative to adding each amino acid separately in thesynthesis, some may be coupled to one another prior to addition to thesolid-phase synthesizer. The selection of an appropriate couplingreagent is within the skill of the art. Particularly suitable as acoupling reagent is N,N'-dicyclohexyl carbodiimide ordiisopropylcarbodiimide.

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in excess, and the coupling is suitably carried outin a medium of dimethylformamide (DMF) or CH₂ Cl₂ or mixtures thereof.If incomplete coupling occurs, the coupling procedure is repeated beforeremoval of the N-amino protecting group prior to the coupling of thenext amino acid. The success of the coupling reaction at each stage ofthe synthesis may be monitored. A preferred method of monitoring thesynthesis is by the ninhydrin reaction, as described by Kaiser et al.,Anal. Biochem, 34: 595 (1970). The coupling reactions can be performedautomatically using well-known methods, for example, a BIOSEARCH 9500™peptide synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting groupsis suitably accomplished simultaneously or stepwise. When the resinsupport is a chloromethylated polystyrene resin, the bond anchoring thepeptide to the resin is an ester linkage formed between the freecarboxyl group of the C-terminal residue and one of the manychloromethyl groups present on the resin matrix. It will be appreciatedthat the anchoring bond can be cleaved by reagents that are known to becapable of breaking an ester linkage and of penetrating the resinmatrix.

One especially convenient method is by treatment with liquid anhydroushydrogen fluoride. This reagent not only will cleave the peptide fromthe resin but also will remove all protecting groups. Hence, use of thisreagent will directly afford the fully deprotected peptide. When thechloromethylated resin is used, hydrogen fluoride treatment results inthe formation of the free peptide acids. When the benzhydrylamine resinis used, hydrogen fluoride treatment results directly in the freepeptide amines. Reaction with hydrogen fluoride in the presence ofanisole and dimethylsulfide at 0C for one hour will simultaneouslyremove the side-chain protecting groups and release the peptide from theresin.

When it is desired to cleave the peptide without removing protectinggroups, the protected peptide-resin can undergo methanolysis to yieldthe protected peptide in which the C-terminal carboxyl group ismethylated. The methyl ester is then hydrolyzed under mild alkalineconditions to give the free C-terminal carboxyl group. The protectinggroups on the peptide chain then are removed by treatment with a strongacid, such as liquid hydrogen fluoride. A particularly useful techniquefor methanolysis is that of Moore et al., Peptides, Proc. Fifth Amer.Pept. Symp., M. Goodman and J. Meienhofer, Eds., (John Wiley, N.Y.,1977), p. 518-521, in which the protected peptide-resin is treated withmethanol and potassium cyanide in the presence of crown ether.

Another method for cleaving the protected peptide from the resin whenthe chloromethylated resin is employed is by ammonolysis or by treatmentwith hydrazine. If desired, the resulting C-terminal amide or hydrazidecan be hydrolyzed to the free C-terminal carboxyl moiety, and theprotecting groups can be removed conventionally.

It will also be recognized that the protecting group present on theN-terminal α-amino group may be removed preferentially either before orafter the protected peptide is cleaved from the support.

Purification of the polypeptides of the invention is typically achievedusing conventional procedures such as preparative HPLC (includingreversed-phase HPLC) or other known chromatographic techniques such asgel permeation, ion exchange, partition chromatography, affinitychromatography (including monoclonal antibody columns), orcountercurrent distribution.

The peptides of this invention may be stabilized by polymerization. Thismay be accomplished by crosslinking monomer chains with polyfunctionalcrosslinking agents, either directly or indirectly, throughmulti-functional polymers. Ordinarily, two substantially identicalpolypeptides are crosslinked at their C- or N-termini using abifunctional crosslinking agent. The agent is used to crosslink theterminal amino and/or carboxyl groups. Generally, both terminal carboxylgroups or both terminal amino groups are crosslinked to one another,although by selection of the appropriate crosslinking agent the alphaamino group of one polypeptide is crosslinked to the terminal carboxylgroup of the other polypeptide. Preferably, the polypeptides aresubstituted at their C-termini with cysteine. Under conditions wellknown in the art a disulfide bond can be formed between the terminalcysteines, thereby crosslinking the polypeptide chains. For example,disulfide bridges are conveniently formed by metal-catalyzed oxidationof the free cysteines or by nucleophilic substitution of a suitablymodified cysteine residue. Selection of the crosslinking agent willdepend upon the identities of the reactive side chains of the aminoacids present in the polypeptides. For example, disulfide crosslinkingwould not be preferred if cysteine was present in the polypeptide atadditional sites other than the C-terminus. Also within the scope hereofare peptides crosslinked with methylene bridges.

Suitable crosslinking sites on the peptides, aside from the N-terminalamino and C-terminal carboxyl groups, include epsilon amino groups foundon lysine residues, as well as amino, imino, carboxyl, sulfhydryl, andhydroxyl groups located on the side chains of internal residues of thepeptides or residues introduced into flanking sequences. Crosslinkingthrough externally added crosslinking agents is suitably achieved, e.g.,using any of a number of reagents familiar to those skilled in the art,for example, via carbodiimide treatment of the polypeptide. Otherexamples of suitable multi-functional (ordinarily bifunctional)crosslinking agents are found in the literature.

The peptides of this invention also may be conformationally stabilizedby cyclization. The peptides ordinarily are cyclized by covalentlybonding the N- and C-terminal domains of one peptide to thecorresponding domain of another peptide of this invention so as to formcyclo-oligomers containing two or more iterated peptide sequences, eachinternal peptide having substantially the same sequence. Further,cyclized peptides (whether cyclo-oligomers or cyclo-monomers) arecrosslinked to form 1-3 cyclic structures having from 2 to 6 peptidescomprised therein. The peptides preferably are not covalently bondedthrough α-amino and main-chain carboxyl groups (head to tail), butrather are crosslinked through the side chains of residues located inthe N- and C-terminal domains. The linking sites thus generally will bebetween the side chains of the residues.

Many suitable methods per se are known for preparing mono-orpoly-cyclized peptides as contemplated herein. Lys/Asp cyclization hasbeen accomplished using Na-Boc-amino acids on solid-phase support withFmoc/9-fluorenylmethyl (OFm) side-chain protection for Lys/Asp; theprocess is completed by piperidine treatment followed by cyclization.

Glu and Lys side chains also have been crosslinked in preparing cyclicor bicyclic peptides: the peptide is synthesized by solid-phasechemistry on a p-methylbenzhydrylamine resin. The peptide is cleavedfrom the resin and deprotected. The cyclic peptide is formed usingdiphenylphosphorylazide in diluted methylformamide. For an alternativeprocedure, see Schiller et al., Peptide Protein Res., 25: 171-177(1985). See also U.S. Pat. No. 4,547,489.

Disulfide crosslinked or cyclized peptides are generated by conventionalmethods. The method of Pelton et al. (J. Med. Chem., 29: 2370-2375(1986)) is suitable, except that a greater proportion of cyclo-oligomersare produced by conducting the reaction in more concentrated solutionsthan the dilute reaction mixture described by Pelton et al., for theproduction of cyclo-monomers. The same chemistry is useful for synthesisof dimers or cyclo-oligomers or cyclo-monomers. Also useful arethiomethylene bridges. Lebl and Hruby, Tetrahedron Letters, 25:2067-2068 (1984). See also Cody et al., J. Med. Chem., 28: 583 (1985).

The desired cyclic or polymeric peptides are purified by gel filtrationfollowed by reversed-phase high-pressure liquid chromatography or otherconventional procedures. The peptides are sterile filtered andformulated into conventional pharmacologically acceptable vehicles.

The starting materials required for the processes described herein areknown in the literature or can be prepared using known methods and knownstarting materials.

If in the peptides being created carbon atoms bonded to fournonidentical substituents are asymmetric, then the compounds may existas diastereoisomers, enantiomers, or mixtures thereof. The synthesesdescribed above may employ racemates, enantiomers, or diastereomers asstarting materials or intermediates. Diastereomeric products resultingfrom such syntheses may be separated by chromatographic orcrystallization methods. Likewise, enantiomeric product mixtures may beseparated using the same techniques or by other methods known in theart. Each of the asymmetric carbon atoms, when present, may be in one oftwo configurations (R or S) and both are within the scope of the presentinvention.

The compounds described in this invention may be isolated as the freeacid or base or converted to salts of various inorganic and organicacids and bases. Such salts are within the scope of this invention.Examples of such salts include ammonium, metal salts like sodium,potassium, calcium, and magnesium; salts with organic bases likedicyclohexylamine, N-methyl-D-glucamine, and the like; and salts withamino acids like arginine or lysine. Salts with inorganic and organicacids may be likewise prepared, for example, using hydrochloric,hydrobromic, sulfuric, phosphoric, trifluoroacetic, methanesulfonic,malic, maleic, fumaric acids, and the like. Non-toxic andphysiologically compatible salts are particularly useful, although otherless desirable salts may have use in the processes of isolation andpurification.

A number of methods are useful for the preparation of the saltsdescribed above and are known to those skilled in the art. Examplesinclude reaction of the free-acid or free-base form of the peptide withone or more molar equivalents of the desired acid or base in a solventor solvent mixture in which the salt is insoluble; or in a solvent likewater after which the solvent is removed by evaporation, distillation,or freeze drying. Alternatively, the free-acid or -base form of theproduct may be passed over an ion-exchange resin to form the desiredsalt or one salt form of the product may be converted to another usingthe same general process.

Certain specific schemes that may be appropriate for chemical synthesisof the peptides herein are shown in WO 96/15148 published May 23, 1996,the disclosure of which is incorporated herein by reference.

The compounds of this invention are shown to prevent the interaction ofan IGF with one or more of its binding proteins and thereby agonize IGFaction. It is known to those skilled in the art that there are many usesfor IGFs. Therefore, administration of the compounds of this inventionfor purposes of agonizing an IGF action can have the same effects oruses as administration of an exogenous IGF itself. These uses of IGFinclude the following, which may be additional to or the same as thedisorders as defined above: increasing whole body, bone, and musclegrowth rate in normal and hypopituitary animals; protection of bodyweight and nitrogen loss during catabolic states (such as fasting,nitrogen restriction, elevated corticosteroid levels, and/or diabetes);kidney regeneration; treating peripheral and central nervous system(CNS) degenerative disorders and promoting neuroprotection or repairfollowing CNS damage or injury; treating hypoxia; promotion of woundhealing; cardiac regeneration; reversal of cancer cachexia; inhibitionof angiogenesis; regeneration of the gastrointestinal tract; stimulationof mammary function; counteracting IGF-I-dependent actions of GH such asmetabolic stress, age-related decreases in GH activity, and adult GHdeficiency; treating maturity-onset diabetes; and/or treating a specificIGF deficiency.

Additional and specific disorders for which the compounds herein areuseful include growth disorders such as GH-resistant short stature,GH-insensitivity syndrome, osteoporosis, and catabolic states; disorderswhere treatment requires regeneration of tissues or cells, for example,peripheral nerves and supporting cells, central nervous system cellsincluding nerves and glia, and other cells such as oligodendrocytes,muscle, skin, and bone; heart disorders, e.g., heart ischemia, cardiacmyopathy, and congestive heart disorders; hyperglycemic disorders suchas insulin-dependent and non-insulin-dependent diabetes mellitus andextreme insulin resistance; and renal disorders such as renal failure.These also include stimulation of an anabolic response in elderlyhumans, prevention of catabolic side effects of glucocorticoids,treatment of osteoporosis, stimulation of the immune system, reductionof obesity, acceleration of wound healing, acceleration of bond fracturerepair, treatment of growth retardation, treatment of renal failure orinsufficiency resulting in growth retardation, treatment ofphysiological short stature, including growth-hormone-deficientchildren, treating short stature associated with chronic illness,treatment of obesity and growth retardation associated with obesity,treatment of growth retardation associated with Prader-Willi syndromeand Turner's syndrome, acceleration of the recovery and reduction in thehospitalization of burn patients, treatment of interuterine growthretardation, skeletal dysplasia, hypercortisolism, and Cushingssyndrome, induction of pulsatile growth hormone release, replacement ofgrowth hormone in stressed patients, treatment ofosteochondrodysplasias, Noonans syndrome, schizophrenia, depression,peripheral neuropathy, ALS, depression, Alzheimer's disease, diseases ofdemyelination, multiple sclerosis, and delayed wound healing,stimulation of the immune system, treatment of physcosocia depravation,treatment of pulmonary dysfunction and ventilator dependency,attenuation of protein catabolic response after a major operation,reduction of cachexia and protein loss due to chronic illness such ascancer or AIDS, treatment of hyperinsulinemia including Type II and TypeI diabetes, adjuvant treatment for ovulation induction, stimulation ofthymic development and prevention of the age-related decline of thymicfunction, treatment of immunosuppressed patients, treatment ofbone-marrow transplanted patients, improvement in muscle strength,mobility, diseases of muscle function, muscular dystrophy, maintenanceof skin thickness, and metabolic homeostasis, enhancement of renalfunction and homeostasis including acute and chronic renal failure,stimulation of osteoblasts, bone remodeling, and cartilage growth,stimulation of the immune system, and growth promotion in livestock.Various IGF-I uses are found, for example, in WO 94/04569; WO 96/33216;and Bondy, Ann Intern. Med., 120: 593-601 (1994). All these are includedin the definition of "disorder."

In one example, the compounds can be administered to commerciallyimportant mammals such as swine, cattle, sheep, and the like toaccelerate and increase their rate and extent of growth and theefficiency of their conversion of feed into body tissue. The compoundscan be administered in vivo to adults and children to stimulate IGFaction.

The compounds of this invention may be administered to the mammal by anysuitable technique, including oral, parenteral (e.g., intramuscular,intraperitoneal, intravenous, or subcutaneous injection or infusion, orimplant), nasal, pulmonary, vaginal, rectal, sublingual, or topicalroutes of administration, and can be formulated in dosage formsappropriate for each route of administration. The specific route ofadministration will depend, e.g., on the medical history of the patient,including any perceived or anticipated side effects using the compound,the type of compound being administered, and the particular disorder tobe corrected. Most preferably, the administration is orally or bycontinuous infusion (using, e.g., slow-release devices or minipumps suchas osmotic pumps or skin patches), or by injection (using, e.g.,intravenous or subcutaneous means).

The compound to be used in the therapy will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with the compound), the site of delivery, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The "effective amounts" of the compoundfor purposes herein are thus determined by such considerations and mustbe amounts that result in bioavailability of the drugs to the mammal andthe desired effect.

If a small-molecule antagonist is used as an IGF agonist, it may havecyclical effects and require, for efficacy, an administration regimenappropriate thereto, the variable concentration of IGFBP-l in bloodbeing an example. Jones and Clemmons, supra. For a peptide, a preferredadministration is a chronic administration of about two times per dayfor 4-8 weeks to reproduce the effects of IGF-I. Although injection ispreferred, chronic infusion may also be employed using an infusiondevice for continuous subcutaneous (SC) infusions. A small peptide maybe administered orally. An intravenous bag solution may also beemployed. The key factor in selecting an appropriate dose for diabetesis the result obtained, as measured by decreases in blood glucose so asto approximate the normal range, or by other criteria for measuringtreatment of diabetes as are deemed appropriate by the medicalpractitioner.

As a general proposition, the total pharmaceutically effective amount ofthe IGF agonist compound administered parenterally per dose will be in arange that can be measured by a dose-response curve. For example, IGFsbound to IGFBPs or in the blood can be measured in body fluids of themammal to be treated to determine the dosing. Alternatively, one canadminister increasing amounts of the IGF agonist compound to the patientand check the serum levels of the patient for IGF-I and IGF-II. Theamount of IGF agonist to be employed can be calculated on a molar basisbased on these serum levels of IGF-I and IGF-II. See the examples belowon displacement of IGF-I tracer from IGFBPs present in human serum.

Specifically, one method for determining appropriate dosing of thecompound entails measuring IGF levels in a biological fluid such as abody or blood fluid. Measuring such levels can be done by any means,including RIA and ELISA. After measuring IGF levels, the fluid iscontacted with the compound using single or multiple doses. After thiscontacting step, the IGF levels are re-measured in the fluid. If thefluid IGF levels have fallen by an amount sufficient to produce thedesired efficacy for which the molecule is to be administered, then thedose of the molecule can be adjusted to produce maximal efficacy. Thismethod may be carried out in vitro or in vivo. Preferably, this methodis carried out in vivo, i.e., after the fluid is extracted from a mammaland the IGF levels measured, the compound herein is administered to themammal using single or multiple doses (that is, the contacting step isachieved by administration to a mammal) and then the IGF levels areremeasured from fluid extracted from the mammal.

Another method for determining the amount of a particular IGFBP or theamount of the compound bound to a particular IGFBP in a biological fluidso that dosing of the compound can be adjusted appropriately involves:

(a) contacting the fluid with 1) a first antibody attached to asolid-phase carrier, wherein the first antibody is specific for epitopeson the IGFBP such that in the presence of antibody the IGF binding sitesremain available on the IGFBP for binding to the compound, therebyforming a complex between the first antibody and the IGFBP; and 2) thecompound for a period of time sufficient to saturate all available IGFbinding sites on the IGFBP, thereby forming a saturated complex;

(b) contacting the saturated complex with a detectably labeled secondantibody which is specific for epitopes on the compound which areavailable for binding when the compound is bound to the IGFBP; and

(c) quantitatively analyzing the amount of the labeled second antibodybound as a measure of the IGFBP in the biological fluid, and thereforeas a measure of the amount of the compound bound. This technique can beexpanded to include a diagnostic use whereby the compound isadministered to a mammal to displace an IGF from a specific IGFBP forwhich the compound has affinity, such as IGFBP-1 or IGFBP-3, andmeasuring the amount that is displaced.

The quantitative technique mentioned above using antibodies, called theligand-mediated immunofunctional method (LIFA), is described fordetermining the amount of IGFBP by contact with IGF in U.S. Pat. No.5,593,844, and for determining the amount of GHBP by contact with GH inU.S. Pat. No. 5,210,017. The disclosures of these patents areincorporated herein by reference regarding antibodies and othermaterials and conditions that can be used in the assay.

Another method for determining dosing is to use antibodies to the IGFagonist or another detection method for the IGF agonist in the LIFAformat. This would allow detection of endogenous or exogenous IGFs boundto IGFBP and the amount of IGF agonist bound to the IGFBP.

Another method for determining dosing would be to measure the level of"free" or active IGF in blood. For some uses the level of "free" IGFwould be a suitable marker of efficacy and effective doses or dosing.

For example, one method is described for detecting endogenous orexogenous IGF bound to an IGF binding protein or the amount of acompound that binds to an IGF binding protein and does not bind to ahuman IGF receptor bound to an IGF binding protein or detecting thelevel of unbound IGF in a biological fluid. This method comprises:

(a) contacting the fluid with 1) a means for detecting the compound thatis specific for the compound (such as a first antibody specific forepitopes on the compound) attached to a solid-phase carrier, such thatin the presence of the compound the IGF binding sites remain availableon the compound for binding to the IGF binding protein, thereby forminga complex between the means and the IGF binding protein; and 2) thecompound for a period of time sufficient to saturate all available IGFbinding sites on the IGF binding protein, thereby forming a saturatedcomplex;

(b) contacting the saturated complex with a detectably labeled secondmeans which is specific for one or more sites on the IGF binding protein(such as a second antibody specific for epitopes on the IGFBP) which areavailable for binding when the compound is bound to the IGF bindingprotein; and

(c) quantitatively analyzing the amount of the labeled means bound as ameasure of the IGFBP in the biological fluid, and therefore as a measureof the amount of bound compound and IGF binding protein, bound IGF andIGF binding protein, or active IGF present in the fluid.

Given the above methods for determining dosages, and assuming dosingshares at least some of the characteristics demonstrated in Example 11for IGF-I, in general, the amount of IGF agonist compound that may beemployed can be estimated. An orally active small IGF agonist would havea molecular weight of approximately 500 daltons, compared to 7500daltons for IGF-I and IGF-II. Assuming the IGF agonist is 16-fold lessable to bind to IGFBPs than IGF-I or IGF-II, then equal weights of IGF-Ior IGF-II and these molecules could be equally effective, so that dosesfrom about 10 μg/kg/day to 200 μg/kg/day might be used, based on kg ofpatient body weight, although, as noted above, this will be subject to agreat deal of therapeutic discretion.

A further method is provided to estimate the distribution of IGFs onspecific IGFBPs, e.g., on IGFBP-1 or IGFBP-3 using the LIFA format.

The compound is suitably administered by a sustained-release system.Suitable examples of sustained-release compositions includesemi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules. Sustained-release matrices include polylactides(U.S. Pat. No. 3,773,919; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biololymers, 22, 547-556 (1983),poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res., 15: 167-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982)),ethylene vinyl acetate (Langer et al., supra), orpoly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also include a liposomally entrapped compound. Liposomescontaining the compound are prepared by methods known per se: DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. U.S.A., 82: 3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. U.S.A., 77: 4030-4034(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;Japanese Pat. Appln. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545;and EP 102,324. Ordinarily, the liposomes are of the small (from orabout 200 to 800 Angstroms) unilamellar type in which the lipid contentis greater than about 30 mol. percent cholesterol, the selectedproportion being adjusted for the most efficacious therapy.

PEGylated peptides having a longer life can also be employed, based on,e.g., the conjugate technology described in WO 95/32003 publishedNovember 30, 1995.

For parenteral administration, in one embodiment, the compound isformulated generally by mixing each at the desired degree of purity, ina unit dosage injectable form (solution, suspension, or emulsion), witha pharmaceutically, or parenterally, acceptable carrier, i.e., one thatis non-toxic to recipients at the dosages and concentrations employedand is compatible with other ingredients of the formulation. Forexample, the formulation preferably does not include oxidizing agentsand other compounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the compounduniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, a buffered solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; glycine; amino acids such as glutamic acid,aspartic acid, histidine, or arginine; monosaccharides, disaccharides,and other carbohydrates including cellulose or its derivatives, glucose,mannose, trehalose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; counter-ions such as sodium;non-ionic surfactants such as polysorbates, poloxamers, or polyethyleneglycol (PEG); and/or neutral salts, e.g., NaCl, KCl, MgCl₂, CaCl₂, etc.

The compound is typically formulated in such vehicles at a pH of from orabout 4.5 to 8. It will be understood that use of certain of theforegoing excipients, carriers, or stabilizers will result in theformation of salts of the compound. The final preparation may be astable liquid or lyophilized solid.

Typical formulations of the peptide or oral secretagogues aspharmaceutical compositions are discussed below. About 0.5 to 500 mg ofthe compound or mixture of compounds, as the free-acid or -base form oras a pharmaceutically acceptable salt, is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., as called for by acceptedpharmaceutical practice. The amount of active ingredient in thesecompositions is such that a suitable dosage in the range indicated isobtained.

Typical adjuvants which may be incorporated into tablets, capsules, andthe like are a binder such as acacia, corn starch, or gelatin; anexcipient such as microcrystalline cellulose; a disintegrating agentlike corn starch or alginic acid; a lubricant such as magnesiumstearate; a sweetening agent such as sucrose or lactose; a flavoringagent such as peppermint, wintergreen, or cherry. When the dosage formis a capsule, in addition to the above materials, it may also contain aliquid carrier such as a fatty oil. Other materials of various types maybe used as coatings or as modifiers of the physical form of the dosageunit. A syrup or elixir may contain the active compound, a sweetenersuch as sucrose, preservatives like propyl paraben, a coloring agent,and a flavoring agent such as cherry. Sterile compositions for injectioncan be formulated according to conventional pharmaceutical practice. Forexample, dissolution or suspension of the active compound in a vehiclesuch as water or naturally occurring vegetable oil like sesame, peanut,or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or thelike may be desired. Buffers, preservatives, antioxidants, and the likecan be incorporated according to accepted pharmaceutical practice.

The compound to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticcompositions generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The compound ordinarily will be stored in unit or multi-dose containers,for example, sealed ampules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-mL vials are filled with 5 mL ofsterile-filtered 1% (w/v) aqueous solution of compound, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized compound using bacteriostaticWater-for-Injection.

Combination therapy with the IGF agonist compound herein and one or moreother appropriate reagents that increase total IGF in the blood orenhance the effect of the IGF agonist is also part of this invention.These reagents generally allow the IGF agonist compound herein torelease the generated IGF, and include growth-promoting agents.

Growth-promoting agents for this purpose include, but are not limitedto, GH secretagogues that promote the release of endogenous GH inmammals to increase concentrations of the IGF in the blood. Examplesinclude TRH, diethylstilbestrol, theophylline, enkephalins, E seriesprostaglandins, peptides of the VIP-secretin-glucagon-GRF family, andother GH secretagogues such as GHRP-6, GHRP-1 as described in U.S. Pat.No. 4,411,890, and benzo-fused lactams such as those disclosed in U.S.Pat. No. 5,206,235. See also, e.g., WO 96/15148 published May 23, 1996.Other growth-promoting agents include GHRPs, GHRFs, GH, and theiranalogs. For example, GHRPs are described in WO 95/17422 and WO 95/17423both published Jun. 29, 1995; Bowers, J. Pediatr. Endocrinol., 6: 21-31(1993); and Schoen et al., Annual Reports in Medicinal Chemistry, 28:177-186 (1993). GHRFs and their analogs are described, for example, inWO 96/37514 published Nov. 28, 1996.

Additionally, GHRH, any of the IGFBPs, long-acting GH, GH plus GHBP,insulin, or a hypoglycemic agent can be employed in conjunction with theIGF agonist compound herein for this purpose. In addition, IGF-I orIGF-II or an IGF with an IGFBP such as IGF-I complexed to IGFBP-3 canalso be employed with the IGF agonist compound herein. For example,pharmaceutical compositions containing IGF-I and IGFBP in a carrier asdescribed in WO 94/16723 published Aug. 4, 1994 can be used inconjunction with the compound. The entities can be administeredsequentially or simultaneously with the IGF agonist compound. Inaddition, other means of manipulating IGF status, such as regimens ofdiet or exercise, are also considered to be combination treatments aspart of this invention.

If insulin is also administered, it can be any formulation of insulin,but is preferably NPH insulin, and the dose of NPH insulin is from orabout 5 to 50 units/injection (i.e., from or about 0.2 to 2 mg) twice aday subcutaneously. For a combination of insulin and the compound, theratio of NPH insulin to compound in this formulation by weight isgenerally from or about 10:1 to 1:50, preferably from or about 1:1 to1:20, more preferably from or about 1:1 to 1:10, still more preferably,from or about 1:1 to 1:5, and most preferably from or about 1:1 to 1:3.

Furthermore, the formulation is suitably administered along with aneffective amount of a hypoglycemic agent such as a sulfonylurea. Thehypoglycemic agent is administered to the mammal by any suitabletechnique including parenterally, intranasally, orally, or by any othereffective route. Most preferably, the administration is by the oralroute. For example, MICRONASE™ tablets (glyburide) marketed by Upjohn in1.25-, 2.5-, and 5-mg tablet concentrations are suitable for oraladministration. The usual maintenance dose for Type II diabetics, placedon this therapy, is generally in the range of from or about 1.25 to 20mg per day, which may be given as a single dose or divided throughoutthe day as deemed appropriate. Physician's Desk Reference, 2563-2565(1995). Other examples of glyburide-based tablets available forprescription include GLYNASE™ brand drug (Upjohn) and DIABETA™ branddrug (Hoechst-Roussel). GLUCOTROL™ (Pratt) is the trademark for aglipizide (1-cyclohexyl-3-(p-(2-(5-methylpyrazinecarboxamide)ethyl)phenyl)sulfonyl)urea) tablet available in both 5- and10-mg strengths and is also prescribed to Type II diabetics who requirehypoglycemic therapy following dietary control or to patients who haveceased to respond to other sulfonylureas. Physician's Desk Reference,1902-1903 (1995). Other hypoglycemic agents than sulfonylureas, such asthe biguanides (e.g., metformin and phenformin) or thiazolidinediones(e.g., troglitozone), or other drugs affecting insulin action may alsobe employed. If a thiazolidinedione is employed with the compound, it isused at the same level as currently used or at somewhat lower levels,which can be adjusted for effects seen with the compound alone ortogether with the dione. The typical dose of troglitazone (REZULIN™)employed by itself is about 100-1000 mg per day, more preferably 200-800mg/day, and this range is applicable herein. See, for example, Ghazzi etal., Diabetes, 46: 433-439 (1997). Other thiazolidinediones that arestronger insulin-sensitizing agents than troglitazone would be employedin lower doses.

Another aspect of this invention is a composition comprising an IGF anda thiazolidinedione, or a combination of an IGF, a thiazolidinedione,and a compound of this invention. Additionally, a method for effectingglycemic control is provided by administering to a mammal in needthereof an effective amount of an IGF and a thiazolidinedione, or aneffective amount of an IGF, a thiazolidinedione, and the compound ofthis invention. The active agents may be administered to the mammalsequentially or together, whether in the same formulation orconcurrently. Effective amounts are determined by the practitioner asdescribed above and would generally mean an amount the same as or lessthan the amount of IGF that is used to treat the condition in question(for example, from about 10 to about 250 μg/kg/day of IGF-I fordiabetes) and an amount of dione that is known to be useful to treat thecondition in question, or if the three are used, the amount of compoundusing the dosages as determined above.

In addition, the invention contemplates using gene therapy for treatinga mammal, using nucleic acid encoding the IGF agonist compound, if it isa peptide. Generally, gene therapy is used to increase (or overexpress)IGF levels in the mammal. Nucleic acids which encode the IGF agonistpeptide can be used for this purpose. Once the amino acid sequence isknown, one can generate several nucleic acid molecules using thedegeneracy of the genetic code, and select which to use for genetherapy.

There are two major approaches to getting the nucleic acid (optionallycontained in a vector) into the patient's cells for purposes of genetherapy: in vivo and ex vivo. For in vivo delivery, the nucleic acid isinjected directly into the patient, usually at the site where the IGFagonist compound is required. For ex vivo treatment, the patient's cellsare removed, the nucleic acid is introduced into these isolated cells,and the modified cells are administered to the patient either directlyor, for example, encapsulated within porous membranes which areimplanted into the patient. See, e.g. U.S. Pat. Nos. 4,892,538 and5,283,187.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. A commonly used vector for ex vivodelivery of the gene is a retrovirus.

The currently preferred in vivo nucleic acid transfer techniques includetransfection with viral vectors (such as adenovirus, Herpes simplex Ivirus, or adeno-associated virus) and lipid-based systems (useful lipidsfor lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample). In some situations it is desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell-surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell-surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g., capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem., 262: 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA, 87: 3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols, see Anderson et al., Science,256: 808-813 (1992). See also WO 93/25673 and the references citedtherein.

Kits are also contemplated for this invention. A typical kit wouldcomprise a container, preferably a vial, for the IGF agonist compoundformulation comprising IGF agonist compound in a pharmaceuticallyacceptable buffer and instructions, such as a product insert or label,directing the user to utilize the pharmaceutical formulation. The kitoptionally includes a container, preferably a vial, for a GH, a GHRP, aGHRH, a GH secretagogue, an IGF, an IGF complexed to an IGFBP, an IGFBP,a GH complexed with a GHBP, insulin, or a hypoglycemic agent.

Also provided is a method for predicting the relative affinity forbinding to a ligand of a peptide that competes with a polypeptide forbinding to the ligand, which peptide is derived from a phage-displayedlibrary, which method comprises incubating a phagemid clonecorresponding to the peptide with the polypeptide in the presence of theligand, serially diluting the phage, and measuring the degree to whichbinding of the phagemid clone to the ligand is inhibited by the peptide,wherein a phagemid clone that is inhibited only at low phageconcentrations has a higher affinity for the ligand than a phagemidclone that is inhibited at both high and low phage concentrations.Details are provided in Example 7 below. Preferably, the ligand is anIGFBP such as IGFBP-1 or IGFBP-3 and the polypeptide is an IGF.

In another embodiment herein, a method is provided for directingendogenous IGF either away from, or towards, a particular site in amammal comprising administering to the mammal an effective amount of thecompound herein that is specific for an IGFBP that is either prevalentat, or absent from, the site. "Sites" for this purpose include specifictissues or organs such as the heart, or such as the brain viabrain-specific IGFBPs. Prevalence at the site indicates that the IGFBPin question is located at the site and constitutes a substantial orbiologically important portion of the IGFBP at the site. This indicationfollows from the specificity for IGFBP-1 versus IGFBP-3 of the compoundsdemonstrated herein.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citationsmentioned herein are expressly incorporated by reference.

EXAMPLES

To discover the effect of molecules that bind to the IGFBPs but not toIGF receptors, a mutant human IGF-I described by Bayne et al., J. Biol.Chem., supra, was produced by recombinant DNA technology in E. coli.Specifically, the plasmid pIGFMI was designed for the production of anIGF-I mutant with amino acid changes at residues 24 and 31 (Y24L,Y31A),also designated (Leu²⁴,Ala³¹)hIGF-I or IGF-M in these examples. Theplasmid was constructed from a basic backbone of pBR322 (Sutcliffe, ColdSpring Harb. Symp. Quant. Biol., 43: 77-90 (1978)) as described for theconstruction of phGH1. Chang et al., Gene, 55: 189-196 (1987). Thetranscriptional and translational sequences required for the expressionof the gene were provided by the alkaline phosphatase promoter and thetrp Shine-Dalgarno sequence. Chang et al., supra. Additionally, thelambda transcriptional termination sequence is located downstream of thegene. Scholtissek and Grosse, Nucleic Acids Res., 15: 3185 (1987).Secretion of the protein from the cytoplasm to the periplasmic space isdirected by the lamB signal sequence. Clement and Hofnung, Cell, 27:507-514 (1981). The nucleotide and amino acid sequences for the lamBsignal sequence and the IGF-I mutant (Y24L,Y31A) are given in FIG. 1.

The vector fragment for the construction of pIGFMI was isolated bydigestion of p131TGF with XbaI and ClaI. The vector p131TGF contains thesequences for the alkaline phosphatase promoter, the ampicillin andtetracycline resistance markers, and the trp Shine-Dalgarno sequence.The LamB signal sequence and amino acids 1-15 of IGF-I were provided bydigestion of pBKIGF-IIB (U.S. Pat. No. 5,487,980) with XbaI and PstI.This fragment of approximately 120 bp was isolated and pre-ligated tothe following strands of synthetic DNA encoding the amino acid changesY24L and Y31A (codons underlined):

5'-G TTC GTA TGT GGT GAT CGA GGC TTC CTG TTC AAC AAA CCG ACT GGG GCT G3'-ACGT C AAG CAT ACA CCA CTA GCT CCG AAG GAC AAG TTG TTT GGC TGA CCCCGA CCTAG

(SEQ ID NOS:22 and 23, respectively)

The remaining IGF-I coding sequence and the lambda transcriptionalterminator were provided by isolating the approximately 190-bpBamHI-to-ClaI fragment of pBKIGF-IIB. These fragments were then ligatedtogether to construct pIGFMI as illustrated in FIG. 2. The fullnucleotide sequence of pIGFMI is shown in FIGS. 3A-3D. Bayne et al., J.Biol. Chem., supra, described the properties of IGF-I mutants in termsof their binding to IGF binding proteins or to IGF or insulin receptors.This work showed that the tyrosine residues at positions 24, 31, and 60on human IGF-I are important for binding to the Type 1 IGF-I receptor.The mutant (Leu²⁴, Ala³¹)hIGF-I, where two of these tyrosine residuesare mutated, has a half maximal inhibition of ligand binding of 2,000 nMcompared to the affinity of wild-type hIGF-I of 8.7 nM. This indicates arelative reduction in affinity for the Type 1 IGF receptor (derived fromplacental membranes) by about 250-fold of (Leu²⁴, Ala³¹)hIGF-I comparedto wild-type hIGF-I.

The functional effect of mutations in IGF-I on activity in vitro wasalso studied by Bayne et al., J. Biol. Chem., supra.

The assay system used, the incorporation of 3H thymidine into L7 murinefibroblasts, shows a good correlation between the ability of IGF mutantsto bind to the Type 1 receptor and their ability to stimulate DNAsynthesis. This is reflected in the reduction in activity of (Leu²⁴,Ala³¹)hIGF-I by more than 200-fold in this in vitro activity assay.However, the binding of (Leu²⁴, Ala³¹)hIGF-I to human serum IGFBPs issimilar to that seen for wild-type IGF-I. Bayne et al., J. Biol. Chem.,supra. Therefore, in the following examples, (Leu²⁴, Ala³¹)hIGF-I waschosen to be tested in animals. This molecule was chosen because themutant contains only two mutations, because Type 1 IGF receptor bindingis reduced more than 200-fold, and because binding to the IGFBPs islargely maintained.

Example 1 In vitro Activity of (Leu²⁴, Ala³¹)hIGF-I

To test the direct activity of the IGF-I mutant on the IGF receptor, twodifferent assays were employed. A third assay was used to determine ifthe mutant could displace IGF from IGFBPs in a competitive environment.

Assay 1: KIRA for Phosphorylation of the Human Type 1 Receptor

This assay is a direct activity assay for the human Type 1 receptor.When a receptor in the tyrosine kinase family, such as the Type 1 IGFreceptor, is activated, it is phosphorylated on tyrosine residues. Inthis assay cells containing the Type 1 IGF receptor are activated invitro, then disrupted, and antibodies against the receptor are used toprecipitate the IGF receptor. Next, an anti-phosphotyrosine antibody isused to assay the amount of Type 1 IGF receptor that is phosphorylated.If a fixed number of cells is used, then the amount of receptor that isphosphorylated is a direct measure of the activity of a molecule on theType 1 IGF receptor.

A KIRA for the human Type 1 IGF-I receptor was developed using humanMCF-7 cells. This cell line was derived originally from a human breastcancer tumor and is available from the ATCC. The growth of these cellsis known to respond to the addition of IGF-I. When IGF-I is added tothese cells, a dose-related increase in the phosphorylation of the IGF-Ireceptor occurs (FIG. 4). The addition of the IGF mutant did not causeany change in receptor phosphorylation (FIG. 4). There was no indicationof phosphorylation even at very high concentrations of the mutant IGF.

Specifically, the assay is as follows:

Cells

MCF-7, an adherent cell line isolated from a human breastadenocarcinoma, was purchased from American Type Culture Collection(ATCC-HTB 22; American Type Culture Collection, Rockville, Md.). MCF-7cells have been shown to express measurable levels of surface IGF-IR byFACS analysis. For culture passage, the cells were cultured in 150 cm²tissue-culture flasks (Corning Inc, Corning, N.Y.), 1.5×10⁶ /flask for4-7 days. For the assay, cells were detached from the tissue-cultureflasks with PBS/5mM EDTA, quantified, and cultured in flat-bottommicrotiter plates (Falcon 3072, Becton Dickinson Labware, Lincoln Park,N.J.), 2×10⁵ per well, overnight at 37° C. in 5% CO₂.

Media

Cells were grown in F12/DMEM 50:50 prepared in the media facility ofGenentech, Inc. (obtained from Gibco as a custom formulation, Gibco/BRL,Life Technologies, Grand Island, N.Y.). The medium was supplemented with10% FBS (HyClone, Logan, Utah), 25 mM HEPES (Gibco), and 2 mML-glutamine (Gibco).

KIRA-ELISA

MCF7 cells (2×10⁵) in 100 μl medium were added to each well in aflat-bottom, 96-well culture plate and cultured overnight at 37° C. in5% CO₂. The following morning the well supernatants were decanted, andthe plates were lightly blotted on a paper towel. Stimulation media(F12/DMEM 50:50 with 25 mM HEPES and 2.0% BSA) containing eitherexperimental samples or the recombinant hIGF-I standards were then addedto each well. The cells were stimulated at 37° C. for 30 min., the wellsupernatants were decanted, and the plates were once again lightlyblotted on a paper towel. To lyse the cells and solubilize thereceptors, 100 μl of lysis buffer were added to each well. Lysis bufferconsisted of 150 mM NaCl containing 50 mM HEPES (Gibco), 0.5% Triton-X100 (Gibco), 0.01% thimerosol, 30 KIU/ml aprotinin (ICN Biochemicals,Aurora, Ohio), 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoridehydrochloride (AEBSF; ICN Biochemicals), and 2 mM sodium orthovanadate.The plate was then agitated gently on a plate shaker (BellcoInstruments, Vineland, N.J.) for 60 min. at room temperature.

While the cells were being solubilized, an ELISA microtiter plate (NuncMaxisorp, Inter Med, Denmark) coated overnight at 4° C. with thepolyclonal anti-IGF-IR (antibodies to human Type 1 IGF-I receptor,catalogue 3B7, Santa Cruz Biotech, 5.0 μg/ml in PBS, 100 μl/well) wasdecanted, blotted on a paper towel and blocked with 150 μl/well of BlockBuffer (PBS containing 0.5% BSA (Intergen Company, Purchase, N.Y.) and0.01% thimerosol) for 60 min. at room temperature with gentle agitation.After 60 minutes, the anti-IGF-IR-coated plate was washed six times withwash buffer (PBS containing 0.05% Tween-20 and 0.01% thimerosol) usingan automated plate washer (ScanWasher 300, Skatron Instruments, Inc,Sterling, Va.).

The lysate containing solubilized IGF-IR from the cell-culturemicrotiter well was transferred (85 μl/well) to anti-IGF-IR-coated and-blocked ELISA wells and incubated for 2 h at room temperature withgentle agitation. The unbound receptor was removed by washing with washbuffer, and 100 μl of biotinylated antibody 4G10 (anti-phosphotyrosine)diluted to 0.1 μg/ml in dilution buffer (PBS containing 0.5% BSA, 0.05%Tween-20, 5 mM EDTA, and 0.01% thimerosol) was added to each well. Afterincubation for 2 h at room temperature the plate was washed and 100 μlof HRP-conjugated Dextran-streptavidin (Amdex Laboratories) diluted1:50,000 in dilution buffer was added to each well. The plate wasincubated for 30 minutes at room temperature with gentle agitation. Thefree avidin-conjugate was washed away and 100 μl of freshly preparedsubstrate solution (tetramethyl benzidine; TMB, 2-component substratekit; Kirkegard and Perry, Gaithersburg, Md.) was added to each well. Thereaction was allowed to proceed for 10 minutes, after which the colordevelopment was stopped by the addition at 100 μl/well of 1.0 M H₃ PO₄.The absorbance at 450 nm was read with a reference wavelength of 650 nm(Abs₄₅₀), using a vmax plate reader (Molecular Devices, Palo Alto,Calif.) controlled with a MACINTOSH CENTRIS 650™ computer (AppleComputers, Cupertino, Calif.) and SOFTMAX™ software (Molecular Devices).

The standard curve (FIG. 4) was generated by stimulating MCF7 cells with300, 100, 33.3, 11.1, 3.7, 1.2, 0.4, or 0 ng/ml IGF-I as a referencestandard (Genentech, Inc., lot 1189-2 or equivalent). Sampleconcentrations were obtained by interpolation of their absorbance on thestandard curve and are expressed in terms of IGF-I ng/ml activity. Themutant did not phosphorylate the receptor in this assay.

Assay 2: Increase in Cell Number of Mouse 3T3 Cells

The activity of the IGF mutant was also measured in a bioassay for theactivity of IGF-I on DNA synthesis and cell replication. In this assaymouse 3T3 cells are cultured, and the IGF mutant or IGF-I is added,followed by tritiated ³ H-thymidine. The amount of 3H-thymidineincorporated is a measure of the replication of DNA and thus of cellreplication. IGF-I increased thymidine incorporation in a dose-relatedmanner (FIG. 5). The IGF mutant showed no activity in this assay evenwhen added at very high concentrations.

Assay 3: In vitro Binding of (Leu²⁴, Ala³¹)hIGF-I to IGFBPs

The mutant (Leu²⁴, Ala³¹)hIGF-I was also tested for its ability todisplace radio-labeled rhIGF-I off the IGFBPs in a competitive bindingassay. This assay involves coating recombinant human (rh) IGFBP-1 orrhIGFBP-3 (160 ng/ml) onto 96-well plates overnight, blocking the platesfor one hour with 0.5% BSA, adding rhIGF-I (25014 0.08 ng/ml) or (Leu²⁴,Ala³¹)hIGF-I for 1 hour, then adding 20,000 cpm of ¹²⁵ I-IGF-I andincubating for 2 hours before washing and counting. FIG. 6 shows thatthe mutant (Leu²⁴, Ala³¹)hIGF-I binds to recombinant IGFBP-1 with anaffinity only several fold different from that of wild-type hIGF-I. FIG.7 shows that the mutant (Leu²⁴, Ala³¹)hIGF-I also binds to recombinantIGFBP-3 with an affinity only several fold different from that ofwild-type hIGF-I.

Conclusion

In two in vitro assays the mutant (Leu²⁴, Ala³¹)hIGF-I showed little orno direct activity on the Type 1 IGF-receptor. Firstly, it did notactivate the human IGF-I receptor, as measured by the phosphorylation ofthe receptor in the KIRA assay. Secondly, it did not stimulate the mouseIGF-I receptor directly, as measured by thymidine uptake into 3T3 cells.Therefore, on the basis of the lack of its activation of the IGFreceptor, the IGF mutant would also be expected to be inactive in vivo.However, the mutant (Leu²⁴, Ala³¹)hIGF-I did show significant binding toIGFBP-1 and IGFBP-3 in vitro. This in vitro data provided the basis fortesting the mutant (Leu²⁴, Ala³¹)hIGF-I in vivo.

Example 2 In vivo Activity of (Leu²⁴, Ala³¹)hIGF-I

Introduction

The mutant (Leu²⁴, Ala³¹)hIGF-I tested in Example 1 in vitro was testedin vivo. Even though the mutant (Leu²⁴, Ala³¹)hIGF-I was inactive invitro, it was hypothesized that molecules of this class (molecules thatare inactive directly on receptors, but capable of binding to IGFBPs)would show some activity in vivo. In the first studies (Leu²⁴,Ala³¹)hIGF-I was given by IV injection to conscious rats and effects onglycemic control were determined.

Methods

Seven-week-old male Wistar rats (240-250 g, Charles Rivers Laboratories,Hollister, Calif.) were anesthetized with KETAMINE/XYLAZINE™ anesthesiaand the right jugular vein was cannulated with a silicone rubber cannulathat was developed for chronic blood sampling. Clark et al., J.Endocrinol., 111: 27-35 (1986). Following a two- to three-day recoveryperiod, two basal blood samples were taken at -10 and -5 minutes, thetest substances administered to the rats by IV injection, and then bloodsamples collected after 5, 10, 20, 30, 45, 60, and 120 minutes, and theplasma was immediately separated by centrifugation. The glucose andinsulin concentrations were subsequently determined either by a coupledhexokinase procedure using a Chem 1A serum chemistry analyzer or, in thelater case, a rat insulin RIA kit (Linco Research, Inc., St. Charles,Mo.).

Statistical comparisons were made by an analysis of variance (ANOVA)with a Duncan's Multiple Range test. A p value of <0.05 was consideredas being statistically significant. All data are represented as the mean± SEM.

Results

Study One

Two treatment groups, with five rats per group, were dosed IV witheither (Leu²⁴, Ala³¹)hIGF-I (100 μg) or PBS.

The responses of plasma glucose and plasma insulin to the treatments areshown in FIGS. 8A and 8B, respectively. The data are expressed as apercentage of the values in the pre-treatment blood samples which wereaveraged and set at 100%. One IV injection of (Leu²⁴, Ala³¹)hIGF-I (100μg) caused plasma insulin levels to be immediately (after 5 minutes),dramatically, and significantly (after 10 mins, P<0.001 vs. control)decreased to 25% of that of the control group, and remained depressedfor 60 minutes. This fall in plasma insulin was accompanied by a briefbut statistically significant (after 10 mins, P<0.05 vs. control) fallin blood glucose.

Study Two

The rats in three treatment groups, with 4-5 rats per group, were dosedIV with either (Leu²⁴, Ala³¹)hIGF-I (100/μg) or rhIGF-I (100/μg) and acontrol group was given PBS.

FIGS. 9A and 9B show the responses of plasma glucose (FIG. 9A) andplasma insulin (FIG. 9B) expressed as a percentage of the values in thepre-treatment blood samples which were averaged and set at 100%. Plasmainsulin levels decreased significantly (after 10 mins, p<0.001 vs.control), and remained depressed for 120 minutes in response to the IVinjection of rhIGF-I. In addition, there was a brief but statisticallysignificant fall in blood glucose (after 10 mins, p<0.01 vs. control).These are the expected responses to the injection of rhIGF-I. When(Leu²⁴, Ala³¹)hIGF-I was injected, there was a fall in insulinconcentrations of a similar magnitude and degree of statisticalsignificance (after 10 mins, p<0.001 vs. control) to the fall in insulinin the rats given wild-type rhIGF-I. The insulin levels after (Leu²⁴,Ala³¹)hIGF-I administration remained significantly suppressed to the endof the study (after 120 min., p<0.05 vs. control). There was also asmall but statistically significant fall in blood glucose (after 10min., p<0.05 vs. control).

Conclusion

In two separate experiments, (Leu²⁴, Ala³¹)hIGF-I, a molecule that bindsto IGFBPs with high affinity but binds poorly to IGF receptors, showsIGF-like activity on glycemic response variables when given by IVinjection in a normal rat. This activity could not have been predictedfrom data in the literature. The IGF mutant showed suppressive effectson insulin secretion that were of a similar magnitude to the effect ofwild-type rhIGF-I. The size of this effect, and its similarity to thatseen following administering rhIGF-I itself, were surprising. The fallin glucose levels was not as large as that seen following theadministration of rhIGF-I. This order of responses, a larger effect oninsulin secretion than on blood glucose levels, follows from thesensitivity of these responses to rhIGF-I. A suppression of insulinsecretion is seen at much lower doses of rhIGF-I than are needed tolower blood glucose levels. Furnsinn et al., Endocrinology, 135:2144-2149 (1994).

Example 3 Injections of (Leu²⁴, Ala³¹)hIGF-I Into Diabetic Rats

Introduction

In Example 2 the IGF-I mutant reduced insulin secretion and loweredblood glucose in normal non-diabetic rats. It was unclear if, in thediabetic state, manipulating the endogenous IGF system would lead tosimilar changes in glycemic control. Therefore, an animal model of TypeII diabetes was also chosen to test the glycemic activity of the IGF-Imutant. The animal chosen, the Zucker Rat, is a well-known model ofobesity associated with diabetes in the rat. Stern et al., Proc. Soc.Exp. Biol. Med., 139: 66-69 (1972). A substrain of these rats, theZucker Diabetic Fatty strain (ZDF), is a good model of Type II diabetes,since they become obese and insulin resistant at an early age, withprogressive b-cell failure and then frank diabetes. Johnson et al.,Science, 250: 546-549 (1990). It has been previously shown in ZDF ratsthat IGF-I administration can in the long term slow the onset andseverity of Type II diabetes (WO 96/15148 published May 23, 1996).Therefore, the ZDF rat provides a relevant and sensitive animal model ofType II diabetes to examine the effects of IGF agonist compounds onglycemic control.

In the present example intravenous bolus injections of the IGF mutantwere given and effects on the blood glucose and insulin concentrationswere studied.

Methods

Eighteen 7-week-old male Zucker Diabetic Fatty rats (250-300 g, GeneticModels Inc.) were anesthetized using KETAMINET (62.5 mg/kg)/ROMPUNXYLAZINE™ (12.5 mg/kg) anesthesia. The right jugular vein was cannulatedusing a silicone rubber catheter and the rats were allowed to recover.

Study One

Two days after surgery the rats (5-6/group) were divided into threetreatment groups and given IV via the jugular catheter 200 μl of:

1) the PBS vehicle;

2) IGF mutant (100 μg); or

3) rhIGF-I (100 μg).

The dose of rhIGF-I was chosen as a dose that might cause a small fallin blood glucose levels and in blood insulin levels. The same dose ofIGF mutant was given for comparison.

Study Two

Three days later, using the same rats the study was repeated. The ratsthat were dosed with IGF-I in the initial study received the IGF mutantin the repeat study and vice versa. Control animals received PBS in bothexperiments.

Measurements

Two blood samples were collected from each rat via the jugular cannulaprior to IV dosing with the hormones and then blood samples werecollected after 10, 30, 60, and 120 minutes. The plasma was immediatelyseparated by centrifugation. The glucose concentration was measured by acoupled hexokinase procedure using a Chem 1A serum chemistry analyzer.Insulin concentration was measured using a rat insulin RIA kit (LincoResearch, Inc., St. Charles, Mo.).

Statistical comparisons for each time point were made by an ANOVA with aDuncan's Multiple Range test. A p value of <0.05 was considered as beingstatistically significant. All data are represented as the mean ± SEM,with five or six animals per treatment group.

Results

The results from Study One are shown in FIGS. 10A and 10B and from StudyTwo in FIGS. 11A and 11B. In each Figure the top panel (A) shows thepercentage change in plasma insulin from baseline (set as 100%) and thebottom panel (B) the percentage change in plasma glucose from baseline(set as 100%).

In Study One the IV injection of rhIGF-I caused an immediate, moderate,and statistically significant (p<0.01 vs. control, excipient-treatedrats) fall in blood glucose (FIG. 10B). In comparison, there was a smallrise in blood glucose in the excipient-treated rats. Blood glucose thenrebounded to the level in control animals. A smaller, but statisticallysignificant (P<0.05 vs. control, excipient-treated rats) fall in bloodglucose was also seen after 10 minutes in animals treated with the IGFmutant. Blood glucose then rebounded to control levels. After 120minutes blood glucose drifted to lower levels in the animals treatedwith both rhIGF-I and the IGF mutant. This is suggestive of a long-termeffect of the IGF mutant on blood glucose.

These changes in blood glucose were accompanied by changes in theconcentrations of insulin in the blood. In the control rats bloodinsulin levels were high and the levels were maintained throughout thetwo hours of sampling. The injection of rhIGF-I caused a statisticallysignificant (p<0.01 vs. control, excipient-treated rats), large, andmaintained fall in plasma insulin. Injection of the IGF mutant alsosignificantly (p<0.01, vs. control, excipient-treated rats) suppressedblood insulin concentrations. In this group insulin slowly returned tocontrol levels two hours after the injection.

In Study Two (FIGS. 11A and 11B) similar results were obtained to thosefound in Study One and shown in FIGS. 10A and 10B. In Study Two an IVinjection of rhIGF-I caused an immediate, moderate, and statisticallysignificant (p<0.01 vs. control, excipient-treated rats) fall in bloodglucose after 10 minutes. Blood glucose then rebounded to the level incontrol animals after 30 minutes. In this study a similar andstatistically significant (p<0.05 vs. control, excipient-treated rats)fall in blood glucose was seen after 10 minutes in animals treated withthe IGF mutant. Blood glucose then rebounded to control levels, thendrifted to lower levels. Once again, this is suggestive of a long-termeffect of the IGF mutant on blood glucose.

The changes in blood glucose were accompanied by changes in the insulinconcentrations in the blood. In the controls blood insulin levels werehigh and were maintained throughout the two hours of sampling. Theinjection of rhIGF-I caused a statistically significant (p<0.01 vs.control excipient-treated rats), large, and maintained fall in plasmainsulin. Injection of the IGF mutant caused a similar and significantfall in blood insulin concentrations (p<0.01, vs. control,excipient-treated rats). In both groups insulin slowly returned towardcontrol levels, but after two hours did not appear to have fullyreturned to pre-injection levels or the levels in control rats.

These experiments show that the acute administration of an IGFagonist 1) reduces insulin levels and 2) reduces blood glucose levels.In the present study only one injection was given, and the delivery wasintravenous. Since one injection of the IGF mutant showed efficacy onglycemic parameters, it would be most likely that multiple injectionswould show similar acute glycemic effects leading to beneficiallong-term cumulative effects. It would also be expected that injectionsgiven subcutaneously would have a similar effect to the intravenousinjections used here, but may show a different time course, due to theirslower absorption from the subcutaneous injection site.

In humans, infusions of rhIGF-I inhibit insulin release at much lowerdoses than those needed to reduce blood glucose (Hartman et al., J.Clin. Invest., 91: 2453-2462 (1993)), and such low-dose euglycemicinfusions of rhIGF-I also rapidly suppress fasting-enhanced pulsatile GHsecretion. Therefore, a fall in insulin is expected to be a sensitivemarker of the release of IGF-I after the administration of a moleculethat prevents the interaction of an IGF with one of its IGFBPs, assumingthat the IGF agonist increases the bioavailability of IGF-I, causingendogenous active IGF-I levels to rise.

In the examples shown herein, using normal animals and diabetic animals,it can be deduced that sufficient IGF-I is activated to inducesignificant falls in blood insulin and blood glucose concentrations.

Example 4 Long-term Administration of (Leu²⁴, Ala³¹)hIGF-I to Hypox Rats

Introduction

The previous set of studies addressed the acute effect of administeringmolecules that preferentially bind to the IGFBPs rather than to IGEreceptors. The short-term effect of the administration of thesemolecules is now shown to cause an IGF-like agonist effect, as shown bya fall in blood glucose and insulin levels.

An important issue that then arises is the longer-term effects ofadministration of the IGF agonist. If the mechanism of these activitiesis a simple displacement of IGF, then it is possible that with time,with long-term exposure, with continual exposure, or with high-doseexposure, the response to the IGF agonist will show tachyphylaxis andthe acute response will diminish. If the mechanism of action is otherthan simple displacement of IGF, then the long-term effect in animals ofan IGF agonist is even less certain. In addition, the short-term studieson glucose regulation do not show the longer-term effects of IGF-I, forexample, the anabolic effects, the differentiative effects, the mitoticeffects, and the effects on organ function. It was therefore importantto administer an IGF agonist long term to animals.

The first model chosen was the hypophysectomized rat. Thehypophysectomized rat is very sensitive to the effects of both GH andIGF-I. Guler et al., Proc. Natl. Acad. Sci. USA, (1988), supra; Clark etal., Endocrine, 3: 297-304 (1995). However, as endogenous IGF-I levelsare very low in hypophysectomized rats (perhaps only 10% of normal), itwas possible that the IGF mutant might not show activity by itself.Therefore, a group of hypophysectomized rats were also given recombinanthuman GH. GH treatment in hypophysectomized rats raises the levels ofIGF-I in blood (Guler et al., Proc. Natl. Acad. Sci. USA, (1988), supra;Clark et al., Endocrine, supra), and it was reasonable to assume thatthis increased blood level of IGF-I might be activated by theco-administration of the IGF mutant.

Methods

Young female SD rats were hypophysectomized at Taconic Laboratories anddelivered several days later. The rats were then weighed frequently andthose whose weight increased or decreased by more than 7 grams between 2and 3 weeks following hypophysectomy were excluded from the study.Twenty-five rats were grouped randomly into five groups based on theirbody weight, and then randomly assigned to cages, with four rats percage. The animals were allowed ad libitum access to food and water andhoused in a room controlled for temperature and lighting.

Experimental Groups

1) Excipient pump, excipient injections;

2) (Leu²⁴, Ala³¹)hIGF-I (10 μg/day, by SC minipump), excipientinjections;

3) (Leu²⁴, Ala³¹)hIGF-I (50 μg/day, by SC minipump), excipientinjections;

4) Recombinant human GH (NUTROPIN™ brand from Genentech, Inc., 20μg/day, by SC injection, two injections each of 10 μg/day), excipientminipump; or

5) (Leu²⁴, Ala³¹)hIGF-I (50 μg/day, by SC minipump) plus recombinanthuman GH (20 μg/day, by SC injection, two injections each of 10 μg/day).

The rats were dosed for one week.

Hormones

The mutant (Leu²⁴,Ala³¹)hIGF-I was administered by osmotic minipumps(ALZET ₂₀₀₁ ™, Alza, Palo Alto, Calif.), which were placed in asubcutaneous tunnel in the dorsal neck region of the rats while theywere anesthetized with KETAMINET™/XYLAZINE™ anesthesia. The pumps werefilled with solutions so that the calculated daily dose was 50 μg or 10μg, assuming that 24 μl of solution was delivered each day for one week.

Recombinant human GH was administered for one week by twice daily SCinjections of 10 μg in 100 μl volume.

Animals not receiving injections of rhGH received injections of vehicleand animals which were not administered IGF-I had a saline-filled pumpimplanted.

Measurements

Body weights were measured daily in the morning. At the end of the weekof dosing the animals were anesthetized via CO₂ and exsanguinated viacardiac puncture. The remaining blood was allowed to clot and serum wasseparated and stored for further analysis.

Spleen, thymus, heart, liver, kidney, and perirenal fat were removed andweighed. The tibia was removed and fixed in neutral-buffered formalinfor histological measurement of epiphyseal plate width.

Serum chemistries were measured on an automated Chem I analyzer. Seruminsulin concentration was measured using an RIA Kit supplied by Linco,Inc. To measure the IGF-I concentrations the serum was extracted usingacid ethanol and the supernatant diluted to neutrality and assayed forIGF-I by RIA. The concentration of rat IGF-I in serum extract wasmeasured by RIA using a kit from Diagnostic Systems Laboratories, Inc.This rat IGF-I assay does not measure human IGF-I. Human IGF-Iconcentration was measured by RIA at Genentech, Inc.

Tibia were sectioned longitudinally, stained with Toluene Blue, andmounted on microscope slides. The tibial epiphyseal plate width wasmeasured using an ocular micrometer attached to a microscope.

Data were analyzed statistically by analysis of variance using onefactor and two factor ANOVA.

Results and Discussion

Body Weight

FIG. 12 shows the final body weight gains of the animals at the end ofthe study. There was a significant increase in body weight as a resultof hGH injections. This is the expected response in these GH-deficienthypophysectomized rats, which, when treated with excipients, failed togain weight. By itself, the IGF agonist showed only a small amount ofactivity at 50 μg per day. At 10 μg per day the IGF agonist group had amean weight gain that was numerically smaller than that of the controlgroup. However, when the IGF agonist was given along with hGH, the IGFagonist showed an enhancement of the activity of hGH on whole bodygrowth. This is shown in the final body weights in FIG. 12.

Organ Growth

The organ weight data is illustrated for spleen (FIGS. 13A and 13B),thymus (FIGS. 14A and 14B), and heart (FIGS. 15A and 15B). In eachFigure the absolute organ weight is shown in A, the top panel, and theorgan weight expressed as a percentage of body weight is shown in B, thebottom panel.

Absolute spleen weight (FIG. 13A) was increased by both doses of the IGFmutant alone, as it was by hGH. The response to hGH was greater thanthat to the IGF agonist. However, the combination of hGH plus IGFagonist doubled spleen size with evidence of a synergistic effect. Whenthe spleen size was corrected for the growth of the whole body (byexpressing the data as a percent of whole body weight), the IGF agonistagain showed evidence of activity when given alone. However, now thesynergistic effect of the combination of hGH and IGF mutant was moreobvious, as there was a very large response to combination treatment.

Absolute thymus weight (FIG. 14A) was also increased by the high-doseIGF agonist alone. The responses to hGH and to the IGF displacer werealmost equal. The combination of hGH plus IGF agonist also increasedthymus weight. When the thymus size was corrected for the growth of thewhole body (by expressing the data as a percent of whole body weight),the IGF agonist again showed evidence of activity when given alone, withthe response again being similar to that caused by hGH. The combinationof hGH and IGF mutant also increased relative thymic weight.

In the present Example the IGF agonist at both 10- and 50-μg dosescaused a significant increase in the absolute and the relative size ofthe heart (FIGS. 15A and 15B). In comparison, treatment with hGHslightly increased absolute heart weight and caused a fall in relativeheart weight. Treatment with the IGF mutant in combination with hGHgreatly increased the absolute weight of the heart and reversed theGH-induced decline in relative heart weight.

Bone Growth

The epiphyseal plate widths from the five treatment groups in this studyare shown in FIG. 16. The width of the epiphysis in the controlhypophysectomized rats (about 200 microns) is similar to that reportedin the literature in similar rats. Clark et al., Endocrine, 3: 717-723(1995). When given by itself, the (Leu²⁴,Ala³¹)hIGF-I at 50 μg/dayinduced a significant increase in bone growth (P<0.01 vs. control)Low-dose (Leu²⁴,Ala³¹)hIGF-I did not increase bone growth. Incomparison, the injections of rhGH induced a very large increase in bonegrowth, doubling plate width to around 400 μm. The combination of rhGHand (Leu²⁴,Ala³¹)hIGF-I did not cause a further increase in bone growth.However, an epiphyseal plate width of 400 μm is near maximal, making itunlikely that a further increase could occur with combination dosing atthe doses used. Since there was a growth response to (Leu²⁴,Ala³¹)hIGF-Igiven alone, it would be expected that at lower doses of hGH a greatereffect of the combination would be seen than for each agent given alone.

Serum IGF-I Levels

The levels of IGF-I in the blood were measured in two assays: one assaymeasured the amount of rat IGF-I in the blood, another the amount ofhuman IGF-I in the blood. FIG. 17 shows the amount of rat IGF-I (FIG.17A) and the amount of total IGF-I (FIG. 17B) in the blood in the fivetreatment groups. A remarkable aspect of these data (FIG. 17A) is thatthe blood levels of receptor-active IGF-I (endogenous rat IGF-I) arelower in the animals given the IGF agonist, yet these lower blood IGF-Ilevels were associated with marked IGF-like responses in various organsand tissues. Clearly, it is counter-intuitive to observe evidence ofincreased activity of a hormone when the blood levels of the hormone aredecreased.

The assay for total IGF-I (FIG. 17B) measures both rat and human IGF-I.In this assay there was a rise in total IGF-I in the rats given 50 μg ofIGF-I agonist, probably due to the IGF-I agonist binding to bindingproteins and therefore being present in the blood. At the lower dose of10 μg of IGF agonist, there was not a rise in total IGF in the blood.The total IGF in the blood did rise (p<0.05 vs. hGH alone) in the ratsgiven the combination of GH and the IGF agonist, in contrast to thelevel of rat IGF-I, which tended to fall in this treatment group. Thisindicates that when GH generates IGF-I and IGFBPs, there is sparebinding capacity, and that this capacity has been in part filled by theIGF agonist.

Conclusion

It has been previously shown that the combination of GH and IGF-I, whenadministered together to animals, including humans (U.S. Pat. No.5,126,324; Kupfer et al., J. Clin. Invest., 91: 391-396 (1993)), showsgreater activity than either agent alone. However, it was unclear ifadministering an IGF agonist would result in activation of the IGF axisand enhance the effect of GH administration. The present experimentshows that administering compounds that bind tightly to IGFBPs, but bindpoorly to IGF receptors, can enhance the activity of GH.

It has also been reported that the administration of IGF-I to animalsinduces a different pattern of growth to that induced by GH. Inparticular, IGF-I administration causes the spleen, thymus, and kidneyto show marked overgrowth. Guler et al., Proc. Natl. Acad. Sci. USA, 85:4889-4893 (1988); Skottner et al., Endocrinology, 124: 2519-2526 (1989);Clark et al., Endocrine, 3: 297-304 (1995). However, since the wholebody size of the rats treated with IGF mutant alone showed only a smallresponse, it might be expected that there would also be little change inthe size of the organs of the treated animals. This was not the case; itis shown in this Example that the IGF mutant has very large "IGF-like"effects on some of the organs known to be sensitive to IGF-I, i.e., thespleen and thymus.

A very surprising finding in this study was the effect of the treatmentson the size of the heart. In previous studies in hypophysectomized orGH-deficient rats, it had been shown that the administration of IGF-Ihad a very small or no effect on cardiac size. Guler et al., Proc. Natl.Acad. Sci. USA, 85: 4889-4893 (1988); Skottner et al., Endocrinology,124: 2519-2526 (1989); Clark et al., Endocrine, 3: 297-304 (1995).

In view of the efficacy of the IGF mutant as an agonist of IGF-I asshown above, the present invention is expected to have application inthe treatment of a large group of disorders associated with, orcharacterized by, a lack of active IGF-I in the bloodstream.Representative of such disorders are diabetes, obesity, anabolicdisorders, immunologic disorders, cardiac disorders, and renaldisorders, as well as others noted above.

Example 5 Long-term Administration of (Leu²⁴,Ala³¹)hIGF-I to Dwarf Rats

Introduction

The previous Example addressed the long-term effect in hypophysectomizedrats of administering molecules that preferentially bind to the IGFBPsrather than to IGF receptors, represented by (Leu²⁴,Ala³¹)hIGF-I.Hypophysectomized rats have very low serum IGF-I levels and very lowlevels of IGFBPs because they lack pituitary hormones. Fielder et al.,Endocrinology, 137: 1913-1920 (1996). However, despite these low levelsof endogenous proteins, the IGF mutant showed remarkable activity. Thenext model chosen to test the activity of the IGF mutant was theGH-deficient dwarf (dw/dw) rat (Charlton et al., J. Endocr., 119: 51-58(1988)), which shows a growth response to both GH and IGF-I. Skottner etal., Endocrinology, 124: 2519-2526 (1989); Clark et al., Endocrine3:717-727 (1995). The dw/dw rat is not totally GH-deficient, as is thehypophysectomized rat, and thus has higher levels of serum IGF-I and theIGFBPs. Therefore, the dose of the IGF mutant given to the dw/dw ratswas increased, since the blood of the dw/dw rats was expected to containmore IGF binding capacity and more IGF.

In the hypophysectomized rat (Example 4) GH was given to produce greateramounts of IGF and IGFBP, and the IGF mutant tested in the presence ofthis exogenous hormone. In the present Example the addition of exogenousGH was repeated, as was the addition of exogenous IGF-I, to discover theactivity of the IGF mutant in the presence of these exogenouslyadministered hormones. The aim of the study was to discover the effectsof administering a molecule that binds well to the IGFBPs but bindspoorly to IGF receptors.

Methods

Animals

Young female dwarf rats (11-12 wk of age, 115-140 g) were bred byhomozygous mating (Charles Rivers Laboratories) and delivered to theGenentech Animal House where they were housed five per cage onpolystyrene chips and fed a standard animal chow and water ad libitum,and kept in a room of constant humidity and with controlled temperatureand lighting. The animals were weighed, and based on uniformity of bodyweight, 37 of the animals were selected and randomized into treatmentgroups and cages to give six treatment groups of equal initial bodyweights (approximately 120 g).

Experimental Groups

The study consisted of six groups of rats with six or seven rats pergroup.

    ______________________________________                                        1)     Excipient control                                                                              Excipient injections                                     (Excipient pumps)                                                            2) IGF mutant (IGF-M) Excipient injections                                     (120 μg/day by pump)                                                      3) IGF-I Excipient injections                                                  (120 μg/day by pump)                                                      4) IGF-M (120 μg/day) Excipient injections                                  plus IGF-I (120 μg/day)                                                   5) Excipient pumps hGH injections (50 μg/d)                                6) IGF-M hGH injections (50 μg/d)                                           (120 μg/day by pump)                                                    ______________________________________                                         The rats were dosed for eight days.                                      

Hormones

The mutant (Leu²⁴, Ala³¹)hIGF-I and native-sequence recombinant humanIGF-I were administered by osmotic minipumps (ALZET ₂₀₀₁ ™, Alza, PaloAlto, Calif.), which were placed in a subcutaneous tunnel in the dorsalneck region while the rats were anesthetized with KETAMINE/XYLAZINE™anesthesia. The pumps were filled with solutions so that the calculateddaily dose was 120 μg per rat per day (1 mg/kg/day), assuming that thepump delivered as per the manufacturer's description (24 μl of solutionper day).

Recombinant human GH (50 μg/day) was administered for eight days bytwice-daily sc injection, with each injection being of 25 μg in a volumeof 100 μl.

Animals not receiving injections of rhGH received injections of vehicleand animals which were not administered IGF-M had a saline-filled pumpimplanted.

Measurements:

Body weights were measured daily in the morning. After 8 days theanimals were anesthetized using CO₂ and exsanguinated by cardiacpuncture. The remaining blood was allowed to clot and serum wasseparated and stored for further analysis.

Spleen, thymus, heart, liver, kidney, and perirenal fat were removed andweighed. The tibia was removed and fixed in neutral-buffered formalinfor histological measurement of epiphyseal plate width.

Serum chemistries were measured using a TECHNICON CHEM I PLUS™ analyzer.Serum insulin concentration was measured using an RIA kit supplied byLinco, Inc. To measure the IGF-I concentrations the serum was extractedusing acid ethanol and the supernatant diluted to neutrality and assayedfor IGF-I by RIA. The concentration of rat IGF-I in serum extract wasmeasured by RIA using a kit from Diagnostic Systems Laboratories, Inc.Human IGF-I concentration was measured by RIA at Genentech, Inc.

Tibia were sectioned longitudinally, stained with TOLULENE BLUE™ stain,and mounted on microscope slides. The tibial epiphyseal plate width wasmeasured using an ocular micrometer attached to a microscope.

Data were analyzed statistically by analysis of variance usingone-factor ANOVA followed by Duncan's Range Test.

Results and Discussion

Body Weight

FIG. 18 shows the effect on body weight gain of the treatments over the8 days of the study. On day 7 of the study it was decided to continuethe study for one further day, and a new preparation of hGH was made andinjected. The large weight gain between Days 7 and 8 suggests that thispreparation of hGH was different from that used between Days 0 and 7.Therefore, it was believed that the weight gain data for the hGH groupson Day 7 are a better reflection of the overall experiment than those onDay 8. There were significant increases in body weight as a result ofinfusions of IGF-I or injections of hGH, the expected responses in theseGH-deficient dwarf rats. By itself, the IGF agonist showed only a smallamount of growth-promoting activity, with the weight gain onlyapproaching statistical significance at day 6 (excipient 1.3±1.1 g vs.IGF mutant 4.2±0.6 g, p<0.10). However, when the IGF mutant was givenalong with IGF-I, there was initially clear weight gain (Day oneexcipient -0.6±0.8 g, IGF mutant 0.5±0.6 g, IGF-I 1.2±0.4 g, IGF mutantplus IGF-I 3.3±0.8 g, p<0.05 vs. IGF mutant alone and IGF-I alone), butthis response waned with time. In contrast, the difference between hGHalone and hGH plus the IGF mutant increased with time, reachingstatistical significance by Day 5 (hGH alone 12.4±0.9 g vs. hGH plus IGFmutant 15.5±0.6 g, p<0.05). Therefore, the IGF mutant enhanced theactivity of hGH on whole body growth. These effects are shown in thegrowth curves and the final body weights (FIG. 18).

Organ Growth

The organ weight data are illustrated for spleen (FIG. 19) and kidney(FIG. 20). In each Figure the absolute organ weight is shown in A (thetop panel) and the organ weight expressed as a percentage of body weightis shown in B (the bottom panel).

The absolute spleen weight (FIG. 19A) tended to increase in all groupstreated with the IGF mutant. For example, the spleen weights inexcipient control rats (308±6 mg) were increased by treatment with theIGF mutant alone to 350±14 mg (p<0.10 vs. excipient). Combining theIGF-M with either IGF-I or hGH treatments also tended to give largerspleens (FIG. 19A). When the spleen weights are expressed as apercentage of body weight, similar increases in spleen size caused bythe IGF mutant were seen (FIG. 19B).

The absolute kidney weight and the relative kidney weight are shown inFIGS. 20A and 20B, respectively. The responses of the kidney to the IGFmutant were similar to those of the spleen. For example, relative kidneyweight was increased by treatment with the IGF mutant, especially whenit was given in combination with IGF-I (IGF-I alone, 0.85±0.3% vs IGF-Iplus IGF mutant 0.92±0.04%, p<0.05).

The retroperitoneal fat depot was removed from the rats at sacrifice andweighed. There was a tendency for the IGF mutant to decrease adiposetissue stores (placebo 0.56±0.09 g, IGF mutant 0.53±0.10 g, IGF-I0.48±0.06 g, and IGF-I plus IGF mutant 0.43±0.05 g).

Serum Chemistries and Hormones

Table I shows data collected from analyses of the sera collected atsacrifice. There was some effect of the various treatments on the bloodglucose levels in the rats, but there were very significant effects onthe insulin levels. The diabetogenic effect of GH was shown by thesignificantly (p<0.05) increased insulin concentrations in GH-treatedrats compared to excipient-treated controls. If the rats were co-treatedwith the IGF mutant, this increase in serum insulin (diabetogenic effectof GH) was prevented (Table I), as the serum insulin level in theco-treatment group was no different from the insulin level in theexcipient control group.

                                      TABLE I                                     __________________________________________________________________________    Serum Measurements in Dwarf Rats                                                    Glucose                                                                             Insulin                                                                              BUN    Creatinine                                                                           AST   CK                                       Group (mg/dl) (ng/ml) (mg/dl) (mg/dl) (U/l) (u/l)                           __________________________________________________________________________    Excipient                                                                           198 ± 25                                                                         0.82 ± 0.04                                                                       19.3 ± 0.6                                                                        0.28 ± 0.02                                                                       137 ± 9                                                                          245 ± 47                              IGF-M 174 ± 18 0.88 ± 0.09 17.8 ± 0.4 0.23 ± 0.03 143 ±                                             8 301 ± 44                            IGF-I 190 ± 24 0.54 ± 0.06 16.0 ± 0.7* 0.22 ± 0.05 128 ±                                            11 225 ± 18                           IGF M + 193 ± 19 0.69 ± 0.07 13.8 ± 0.8* 0.18 ± 0.02* 101                                              ± 2* 138 ± 11*                     IGF-I                                                                         hGH 192 ± 10 1.48 ± 0.29* 15.6 ± 0.7* 0.33 ± 0.02 123 ±                                             13 184 ± 29                           hGH + 202 ± 27 1.03 ± 0.20 15.3 ± 0.6* 0.25 ± 0.02 113 ±                                            9 229 ± 30                            IGF-M                                                                       __________________________________________________________________________     Data are Means ± SEM, *p < 0.05 vs excipient control.                 

The IGF mutant (IGF-M) significantly increased kidney size. Twoindications of serum measurements of renal function, the serumcreatinine and the blood urea nitrogen (BUN) levels, were reduced by theIGF mutant. Table I shows that the creatinine concentration in bloodtended to be reduced by both IGF-I and the IGF mutant when they weregiven alone; however, these effects were not statistically significant.However, the combination of IGF-I plus IGF mutant significantly (p<0.05)reduced serum creatinine, indicative of an improvement in renalfunction. A second measure of renal function, the blood urea nitrogen,was also reduced by IGF-I and by IGF-I plus the IGF mutant, with theresponse to the combination being significantly greater than that toIGF-I alone (p<0.05).

The combination of IGF-I plus IGF mutant decreased the amounts of theenzyme AST (alanine serine transferase) and CK (creatinine kinase) inthe blood compared to the excipient controls (p<0.05). AST is a measureof cardiac damage or function and CK is a measure of skeletal musclefunction. These data could be interpreted as showing beneficial effectsof the IGF mutant on skeletal and cardiac muscle.

Bone Growth

The epiphyseal plate widths were significantly (p<0.05) increased by hGHand by IGF-I treatment, compared to excipient-treated controls. The IGFmutant in all cases increased the mean plate widths compared to theindividual treatments, but these increases did not reach statisticalsignificance.

Serum IGF-I Levels

The levels of IGF-I in the blood were measured in two assays (FIGS. 21Aand 21B). One assay (FIG. 21A) measured the amount of rat IGF-I, andanother assay (FIG. 21B) measured both human and rat IGF-I (total IGF-I)in the blood in the five treatment groups. A remarkable aspect of thesedata (FIG. 21A) is that the blood levels of receptor-active IGF-I(endogenous rat IGF-I) were lower in the animals given the mutant IGF,yet these lower blood IGF-I levels were associated with marked IGF-likeresponses in various organs and tissues. Clearly, it iscounter-intuitive to observe evidence of increased activity of a hormonewhen the blood levels of the hormone are decreased.

The assay for total IGF-I (FIG. 21B) measures both rat and human IGF-I.In this assay there was a rise in total IGF-I in the rats given 150 μgof IGF-I agonist, probably due to the IGF-I agonist binding to bindingproteins and therefore being present in the blood. The total IGF levelin the blood did rise (p<0.05 vs. IGF-I alone or hGH alone) in the ratsgiven the combination of either IGF-I or GH and the IGF agonist, incontrast to the level of rat IGF-I, which fell in these combinationtreatment groups. This indicates that when GH generates IGF and IGFBPs,there is spare binding capacity, and that this capacity has been in partfilled by the IGF agonist.

Conclusion

These long-term data in the GH-deficient dwarf rat confirm and extendthe long-term data collected in hypophysectomized rats; the IGF mutantproduced anabolic and growth-promoting effects. In addition, thisexperiment confirms that the IGF mutant shows growth-promoting activitywhen administered with GH. Further, when the IGF mutant was given alongwith IGF-I to the dwarf rats, the IGF mutant could increase the activityof the administered IGF-I. Therefore, the present experiment shows thatthe long-term administration of compounds that bind tightly to IGFBPs,but bind poorly to IGF receptors, can enhance the activity of endogenousIGF-I, exogenously administered GH, and exogenously administered IGF-I.

In this Example several important findings were made which extend thediscoveries made in the hypophysectomized rat.

Firstly, the increased size of the kidney in the rats treated with theIGF mutant was accompanied by a fall in the concentration of creatinineand blood urea nitrogen in the blood. These falls in blood metabolitesare a hallmark of an increased functioning of the kidney. Additionalevidence of a functional effect on cardiac and skeletal muscle was alsoobtained. Evidence of a decrease in fat mass was obtained using the IGFmutant. This indicates uses for IGF agonists in controlling bodycomposition, especially obesity. See, for example, U.S. Pat. No.5,597,797 on use of GH and IGF-I to prevent or treat obesity.

Secondly, the administration of GH significantly increased the bloodconcentrations of insulin, while the co-administration of the IGF mutantprevented this rise in insulin. Therefore, this well-known diabetogeniceffect of GH could be reversed by the co-administration of the IGFmutant.

Thirdly, these responses to the IGF mutant occurred in the presence of afall in the blood concentrations of "active" rat IGF-I.

Therefore, long-term infusions of the IGF mutant showed multipleactivities in 1) hypophysectomized rats, which have no detectable GH intheir blood and very low IGF-I concentrations, and 2) dwarf rats, whichhave low GH and low IGF-I levels in their blood.

Example 6 Long-Term Administration of (Leu²⁴,Ala³¹)hIGF-I to DiabeticRats

In this Example an animal model of diabetes was treated long-term withthe IGF mutant. The animal chosen was a Type II diabetic rat (the ZuckerDiabetic Fatty (ZDF) rat). This rat has relatively normal pituitaryfunction (in terms of GH secretion) and relatively normal serum IGF-Iconcentrations. Therefore, this animal model differs from the twoprevious rat models, which have a complete lack of GH (hypophysectomizedrat) or a clear deficiency of GH (the dw/dw rat) and are both IGFdeficient. In addition, in this Example, the mode of administration ofthe IGF mutant was altered from infusions (used in the two previousExamples) to multiple injections. Furthermore, the dose of the IGFmutant was increased because, as explained above, the ZDF rats are notas IGF deficient as the hypophysectomized or dw/dw rats and thereforehave a larger amount of active IGF to be released. The main endpointsused to measure efficacy in the previous Examples were measures of bodygrowth, while in the present Example the major endpoints were bloodglucose and insulin concentrations, measures of the diabetic state ofthe animals.

In the earlier Examples it was shown that the acute intravenousadministration of one injection of an IGF agonist 1) reduced insulinlevels and 2) reduced blood glucose levels. Since one injection of theIGF agonist showed efficacy on glycemic parameters and long-terminfusions showed efficacy (without tachyphylaxis on growth parameters),it was hypothesized that multiple injections would each show acuteglycemic effects leading to beneficial long-term cumulative glycemiceffects. It was also hypothesized that injections given subcutaneouslywould show efficacy. Therefore, the long-term study in diabetic ratsdescribed below was initiated

Methods and Experimental Groups

Animals

Obese male Zucker Diabetic Fatty (ZDF) rats (6-7 weeks of age, 225-250g, Genetic Models Inc., Indianapolis, Ind. 46268) were group housed(3/cage) in a room controlled for temperature and lighting and fed thepelleted rat diet specified by the breeders (Purina 5008, 6% fat breederchow) and tap water ad libitum. After three days acclimation, a bloodsample was obtained from the tail vein for measurement of blood glucoseand insulin, which was measured by rat-specific RIA (Linco ResearchInc., St. Charles, Mo.). Animals were randomized into four treatmentgroups and into cages to give groups balance so as to have equivalentinitial blood glucose levels, insulin levels, and body weights.

The experiment consisted of four groups of rats with seven rats pergroup:

    ______________________________________                                        1)  Excipient injections three times a day,                                         2)    Injections of IGF mutant (IGF-M)                                                               (50 μg three times a day),                      3) Injections of IGF-M (150 μg three times a day), and                     4) Injections of rhIGF-I (150 μg three times a day).                     ______________________________________                                    

The injections were each of 100 μl given three times a day.

Hormones

The mutant (Leu²⁴,Ala³¹)hIGF-I was prepared at two concentrations (1.5mg/ml and 0.5 mg/ml) so that with three injections per day two doseswere given (a total of 450 μg/d or 150 μg/d), respectively. rhIGF-I wasprepared at one concentration (1.5 mg/ml) so that with three injectionsper day one dose was given (450 μg/d). A fourth group of rats were giventhe excipient. The injections, each of 100-μl volume, were given at 7am, 1 pm, and 7 pm for 15 days without disrupting the controlled diurnallighting of the vivarium room. Body weights were measured each day at 1pm.

Blood Sampling

Blood samples were collected from non-fasted rats on the evening beforethe first day of dosing and on days 3, 7, and 10. Blood samples (400 μl)on days 3, 7, and 10 were taken by bleeding from a tail vein one hourafter the mid-day injection. The blood was allowed to clot, and serumwas separated and stored before analysis.

A glucose tolerance test was performed on day 14 of the study. Ratsreceived their regular morning injection and then their food waswithdrawn. After a 3-5-hour fast a blood sample (400 μl) was taken viathe tail vein. Rats then received 2 g/kg dextrose (50%) byintraperitoneal (ip) injection. A blood sample (200 μl) was taken viatail vein at 10, 60, and 120 minutes after dextrose administration.Blood was allowed to clot and serum was separated and stored formeasurement of glucose and insulin concentrations. After the glucosetolerance test was completed, animals were re-fed.

On day 15, one hour after the morning injection, rats were asphyxiatedwith carbon dioxide and bled by cardiac puncture. Blood was allowed toclot and serum was separated and frozen. Spleen, thymus, heart, liver,kidney, and perirenal fat were removed, blotted dry, and immediatelyweighed.

Statistical Analysis:

Statistical comparisons for each time point were made by an analysis ofvariance with a Duncan's Multiple Range test. A p value of <0.05 wasconsidered as being statistically significant. All data are representedas the mean ± SEM, with seven animals per treatment group.

Results

The body weight gains plotted against time are shown for the fourdifferent treatment groups in FIG. 22A. On day 13 the body weight gainswere significantly increased vs. control (control, 93.3±2.9 g) for theIGF mutant at 50 μg (100±2.1 g, p<0.05 vs. control), for the IGF mutantat 150 μg (105.1±2.3 g, p<0.01 vs. control), and for IGF-I (114.5±1.6 g,p<0.0001 vs. control). The dose-related anabolic effect on body weightof the IGF mutant confirms the data in hypophysectomized and dwarf ratsin the earlier examples.

In these diabetic animals the reason for the gain in body weight couldalso be an indirect effect rather than an anabolic effect because thetreatments improved the diabetic state of the animals. Carlsson et al.,J. Endocrinol., 122: 661 (1989). This interpretation of the data issupported by the illustration in FIG. 22B showing the changes in bloodglucose with time in these same animals. Blood glucose was measuredbefore the injections were commenced, and the Figure shows thepercentage change from this basal level. FIG. 22B shows that the bloodglucose rose steadily in the rats treated with repeated injections ofexcipient to reach 141±21% of basal at day 10, while at this timeinjections of IGF-I (150 μg three times a day) largely prevented(25±10%, p<0.001 vs control) this rise in blood glucose. Injections ofthe IGF mutant at 50 μg (84±20%, p<0.05 vs. control) and the IGF mutantat 150 μg (74±22%, p<0.05 vs. control) also slowed the rise of bloodglucose in these diabetic rats at Day 10. At earlier time points, forexample at day 7, the high-dose IGF mutant and IGF-I were equallyeffective at suppressing the rise in blood glucose.

FIGS. 23A and 23B show the effect of a glucose tolerance test in thesediabetic rats on blood glucose and on insulin concentrations. A glucosetolerance test can be viewed as a simulated meal. The rats were bled andthen given an intraperitoneal injection of glucose at 2 g/kg of bodyweight and were then bled again following the injection. FIG. 23A showsthe change in blood glucose two hours following the glucose challenge.In the diabetic rats treated with injections of excipient, blood glucoserose substantially (105±21 mg/dl). Treatment with IGF-I caused bloodglucose to rise much less (39±12 mg/dl, P<0.05 vs control), whiletreatment with the IGF mutant (150 μg, tid) also blunted this rise(59±11 mg/dl, p<0.1 vs. control).

A possible reason for these beneficial effects of treatment with IGF-1or the IGF mutant is provided by the insulin levels shown in FIG. 23B.In the rats treated with IGF-I more insulin secretion occurred inresponse to the glucose challenge (control, 8.1±0.6 ng/ml vs. IGF-1,12.2±0.7 ng/ml, p<0.01) than it did in the rats treated with IGF mutantat 50 μg (10.2±0.7 ng/ml, p<0.05 vs. control) or 150 μg (10.6±0.6 ng/ml,p<0.05 vs. control). Therefore, long-term treatment with the IGF mutantallowed greater insulin secretion in response to a simulated meal, whichwas associated with an improved ability to dispose of a glucose load.

Conclusions

This long-term study administering the IGF mutant to diabetic rats showsthat blood glucose can be controlled in the long term by manipulatingthe endogenous IGF system. The body weight gain, perhaps an indirectmarker of improved glucose control in diabetic animals, and the directmeasures of glucose control (blood glucose and insulin), show thesurprising efficacy of this class of receptor-inactive molecules inimproving the diabetic state. In the present example multiplesubcutaneous daily injections were given for 15 days with no evidence oftachyphylaxis. In the previous examples infusions of this moleculeproduced maintained anabolic responses. It is therefore reasonable toassume that most routes and patterns of delivery would also show similarefficacy. For example, oral formulations of molecules with longhalf-lives would be expected to be efficacious as wouldshorter-half-life molecules delivered by the oral or other routes.

Example 7 Phage-derived Peptides to Bind IGF-I and Binding Proteins

In the next set of examples, common α-amino acids may be described bythe standard one- or three-letter amino acid code when referring tointermediates and final products. By common α-amino acids is meant thoseamino acids incorporated into proteins under mRNA direction. Standardabbreviations are listed in The Merck Index, 10th Edition, ppMisc-2-Misc-3. Unless otherwise designated, the common α-amino acidshave the natural or "L"-configuration at the alpha-carbon atom. If thecode is preceded by a "D", this signifies the opposite enantiomer of thecommon (α-amino acid. Modified or unusual α-amino acids such asnorleucine (Nle) and ornithine (Orn) are designated as described in U.S.Patent and Trademark Office Official Gazette 1114 TMOG, May 15, 1990.

Based upon the results of experiments using the IGF mutant describedabove, it is predicted that other molecules, such as peptides or smallorganic molecules, which prevent the interaction of an IGF with anIGFBP, and bind poorly or not at all to the IGF-I receptor, shouldincrease active IGF levels in a subject being treated. In addition, itis possible that another class of molecules, in particular peptidic orsmall molecules, might bind IGF-I itself at a site remote from thatinvolved in receptor interactions in such a way as to inhibit or preventthe interaction of IGF-I with the IGFBPs, but not the interaction ofIGF-I with its receptor.

It has been shown that peptides which bind specifically and withmeasurable affinity to target molecules, such as proteins, can beidentified from an initial library of many binding and non-bindingpeptides through binding selections using bacteriophage coat-proteinfusions. Smith, Science, 228: 1315 (1985); Scott and Smith, Science,249: 386 (1990); Cwirla et al., Proc. Natl. Acad. Sci. USA, 8: 309(1990); Devlin et al., Science, 249: 404 (1990); reviewed by Wells andLowman, Curr. Opin. Struct. Biol., 2: 597 (1992); U.S. Pat. No.5,223,409. In addition, both proteins and peptides displayed on phagecan be affinity-enhanced through iterative cycles of mutations,selection, and propagation.

Libraries of peptides differing in sequence at particular residuepositions can be constructed using synthetic oligodeoxynucleotides.Peptides are displayed as fusion proteins with a phage coat protein(such as g3p or g8p) on bacteriophage particles, each of which containsa single-stranded DNA genome encoding the particular peptide variant.After cycles of affinity purification, using an immobilized targetmolecule, individual bacteriophage clones are isolated, and the aminoacid sequence of their displayed peptides is deduced from their DNAsequences.

I. Construction of peptide-phage libraries

To identify a set of peptide molecules having the ability to bind toIGF-I or to an IGF binding protein, such as IGFBP-1 or IGFBP-3, severaldiverse phage libraries of peptides, of length ranging from 18 to 20residues, were constructed. Peptides of this size were chosen in orderto favor the selection of peptides capable of maintaining well-definedstructures in solution. Because natural amino acid peptides of this sizehave a potential sequence diversity of 20¹⁸ -20²⁰ (i.e., 2.6×10²³ to1.0×10²⁶) variants, it is not practical to construct and test all suchvariants. Instead, certain residues were fixed or constant, which mightbe expected to allow or promote stable elements of peptide structuresuch as disulfide bonds or beta-turns, within each peptide.

Structural constraints or frameworks have previously been used forpresentation of peptide libraries on phage and for subsequent,successive enhancement of binding affinities through mutation andselection. Such structured frameworks may favor stable bindingconformations of peptide segments. By analogy, immunoglobulins provide astable (and conserved) structural framework for presentation of adiversity of different peptide loops (CDR's, complementarity-determiningregions) which can bind different antigens.

Used as a template for library constructions was a plasmid, pt4.g8 (DNAsequence shown in FIGS. 24A-C) expressing an antibody-recognizable(gD-tag) peptide fused to g8p of bacteriophage M13. This plasmidcontains single-stranded and double-stranded origins of DNA replication.The phoA promoter and STII secretion-signal sequences are upstream ofthe gD peptide (underlined below), which is followed by a "linker"peptide (double underlined below), and then the g8p of bacteriophageM13:

SGTAMADPNRFRGKDLAGSPGGGSGGGAEGDDPAKAAFNSLQASATEYIGYAWAMVVVIVGATIGIKLFKKFTSKAS (SEQ ID NO:24)

Several random-sequence peptide libraries (Table II) were constructedusing single-stranded template-directed mutagenesis (Kunkel et al.,Methods. Enzymol., 204:125 (1991)), with the oligonucleotides describedbelow.

                  TABLE II                                                        ______________________________________                                        Large Naive Libraries for g8 Display                                            Library  Oligo no. Peptide motif                                            ______________________________________                                        A      HL-300    SGTACX.sub.2 GPX.sub.4 CSLAGSP                                                                SEQ ID NO: 25                                  B HL-301 X.sub.4 CX.sub.2 GPX.sub.4 CX.sub.4 SEQ ID NO: 26                    C HL-302 X.sub.20 SEQ ID NO: 27                                               D HL-303 X.sub.7 CX.sub.4 CX.sub.7 SEQ ID NO: 28                              D HL-304 X.sub.7 CX.sub.5 CX.sub.6 SEQ ID NO: 29                              D HL-305 X.sub.6 CX.sub.6 CX.sub.6 SEQ ID NO: 30                              D HL-306 X.sub.6 CX.sub.7 CX.sub.5 SEQ ID NO: 31                              D HL-307 X.sub.5 CX.sub.8 CX.sub.5 SEQ ID NO: 32                              D HL-308 X.sub.5 CX.sub.9 CX.sub.4 SEQ ID NO: 33                              D HL-309 X.sub.4 CX.sub.10 CX.sub.4 SEQ ID NO: 34                           ______________________________________                                    

A. Beta-turn sequence motif

An example of a peptide of known three-dimensional structure is given byWrighton et al., who selected a peptide agonist for the erythropoietinreceptor (EPO-R) by phage display. Wrighton et al., Science, 273: 458(1996). The peptide GGTYSCHFGPLTWVCKPQGG (SEQ ID NO:35) (having adisulfide bond joining the two Cys residues) forms a dimer of two betahairpins, in the crystallized complex with EPO-R. Livnah et al.,Science, 273: 464 (1996). Although the structure of the unbound form ofthis peptide in solution has not been reported, the beta-turn structureformed by this peptide in complex with EPO-R suggested that similarstructures might be formed by peptides of the form CX₂ GPX₄ C (SEQ IDNO:36).

As one type of structured peptide library, a portion of the gD peptidewas replaced with the motif CX₂ GPX₄ C (SEQ ID NO:37), leaving theupstream and downstream ("flanking") residues unchanged from that of thestarting plasmid. Thus, this library was designed to display on phageparticles the peptide SGTACX₂ GPX₄ CSLAGSP (SEQ ID NO:38), where Xrepresents any of the 20 natural L-amino acids, fused to the linker andg8p described above. This library was constructed using theoligonucleotide HL-300:

5'-GCC TAT GCA TCT GGT ACC GCC TGC NNS NNS GGT CCT NNS NNS NNS NNS TGTTCT CTG GCA GGT TCA CCA G-3' (SEQ ID NO:39),

where N indicates a mixture of the nucleotides A, G, C, and T, and Srepresents a mixture of the nucleotides G and C.

An additional library was constructed to allow for further interactionswithin the peptide and/or with the target proteins by randomizing theflanking sequences as well. This library was constructed with the formX₄ CX₂ GPX₄ CX₄ (SEQ ID NO:40) by using oligonucleotide HL-301:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS TGC NNS NNS GGT CCT NNS NNSNNS NNS TGT NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ ID NO:41).

B. Disulfide-loop motifs

Because many additional peptide conformations might be productive forbinding to a given target protein, it was desirable to test other typesof peptide sequence motifs in phage-displayed libraries. For example, asingle disulfide bond within a small peptide may favor stable structureswhich allow for relatively higher-affinity binding than in unconstrainedstructures. Geysen et al., Mol. Immunol., 23: 709 (1986); Wood et al.,Science, 232: 633 (1986); Oldenburg et al., Proc. Natl. Acad. Sci. USA,89: 5393 (1992); O'Neil et al., Proteins, 14: 509 (1992); McLafferty etal., Gene, 128: 29 (1993); Giebel et al., Biochem., 34: 15430 (1995).Several peptide-phage libraries were therefore constructed, of the formX_(m) CX_(n) CX_(k) (SEQ ID NO:42), where m=4, n=10, and k=4, or wherem=5, n=8-9, and k=4-5, or m=6, n=6-7, and k=5-6, or m=7, n=4-5, andk=6-7 (SEQ ID NOS:28 to 34). In these peptides, a disulfide bond ispredicted to form a stabilizing constraint for peptide conformation.

These peptide libraries (see Table II) were constructed as X₇ CX₄ CX₇(SEQ ID NO:43), using oligonucleotide HL-303:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS NNS TGC NNS NNS NNSNNS TGC NNS NNS NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:44);

X₇ CX₅ CX₆ (SEQ ID NO:45), using oligonucleotide HL-304:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS NNS TGC NNS NNS NNSNNS NNS TGC NNS NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:46);

X₆ CX₆ CX₆ (SEQ ID NO:47), using oligonucleotide HL-305:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS TGC NNS NNS NNS NNSNNS NNS TGC NNS NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:48);

X₆ CX₇ CX₅ (SEQ ID NO:49), using oligonucleotide HL-306:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS TGC NNS NNS NNS NNSNNS NNS NNS TGC NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:50);

X₅ CX₈ CX₅ (SEQ ID NO:51), using oligonucleotide HL-307:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS TGC NNS NNS NNS NNS NNSNNS NNS NNS TGC NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:52);

X₅ CX₉ CX₄ (SEQ ID NO:53), using oligonucleotide HL-308:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS TGC NNS NNS NNS NNS NNSNNS NNS NNS NNS TGC NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:54); and

X₄ CX₁₀ CX₄ (SEQ ID NO:55), using oligonucleotide HL-309:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS TGC NNS NNS NNS NNS NNS NNSNNS NNS NNS NNS TGC NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:56).

C. Unconstrained peptides

Unconstrained libraries (i.e., having no fixed residues within thepeptide) have also yielded specific binding molecules. Scott and Smith,Science, supra; Cwirla et al., Proc. Natl. Acad. Sci. USA, supra; Devlinet al., Science, supra; Kay et al., Gene, 128: 59 (1993). Such librariesmay yield structured peptides, nevertheless, since noncovalentinteractions may still induce structure in the bound and/or unboundforms. An unconstrained peptide library, of the form X₂₀ (SEQ ID NO:57),was constructed using oligonucleotide HL-302:

5'-GCT ACA AAT GCC TAT GCA NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNSNNS NNS NNS NNS NNS NNS NNS NNS NNS GGT GGA GGA TCC GGA GGA G-3' (SEQ IDNO:58).

II. Polyvalent (q8) phage binding selections

The products of random mutagenesis reactions were transformed intoXL1-BLUE™ E. coli cells (Stratagene) by electroporation and amplified bygrowing 15-16 h with M13K07 (Vieira and Messing, Methods Enzymol., 153:3-11 (1987)) or VCSM13 helper phage (Stratagene Corp.). Based uponplating of the initial transformations, the number of transformants perlibrary was approximately 1.8×10⁸ for library HL-300, 7.9×10⁸ forHL-301, 5.0×10⁸ for HL-302, 5.3×10⁸ for HL-303, 5.6×10⁸ for HL-304,5.0×10⁸ for HL-305, 6.3×10⁸ for HL-306, 4.5×10⁸ for HL-307, 1.9×10⁸ forHL-308, and 2.1×10⁸ for HL-309.

IGFBP-1, IGFBP-3, and IGF-I were biotinylated with a 1.5:1 molar ratioof a cleavable biotin reagent, EZ-LINK™ NHS-SS-Biotin (Pierce), toprotein, using the manufacturer's instructions.

The initial selection of peptides for binding to IGFBP-1, IGFBP-3, orIGF-I was carried out using phage pools of approximately 10¹⁰ phage/ml(100 μl total volume). MAXISORP™ 96-well plastic plates (Nunc) werecoated with a solution of 2 μg/mi of NEUTRAVIDIN™ brand avidin (Pierce)in 50 mM sodium carbonate buffer, pH 9.6, overnight at 4° C. TheNEUTRAVIDIN™ solution was then removed, and the plates were incubatedwith a blocking solution of 5 g/l of bovine serum albumin, or 5 g/l ofovalbumin, or 5 g/l of instant milk in 50 mM sodium carbonate buffer,for 1-2 h at room temperature. The blocking solution was then removed,and a solution of biotinylated target protein was added. After 1-2 h atroom temperature, the target solution was removed, and the plates werewashed ten times with PBS/TWEEN™ surfactant (0.05% TWEEN-20™ in PBSbuffer).

Phage from the libraries described above were pooled as follows: pool Aconsisted of HL-300 phage, pool B of HL-301 phage, pool C of HL-302phage, and pool D of phage from the HL-303, HL-304, HL-305, HL-306,HL-307, HL-308, and HL-309 libraries. Phage were added inPBS/TWEEN™/albumin/biotin (PBS/TWEEN™ buffer with 1 μM biotin, 5 g/lbovine serum albumin, or ovalbumin) to wells coated with each target,and with control wells that were coated with NEUTRAVIDIN™ or withalbumin, but not biotinylated target. The phage were allowed to bind5-15 h at room temperature. The plates were then washed ten times withPBS/TWEEN™ buffer.

Phage remaining bound to the plates were eluted by incubating with 50 mMDTT for 1-2 h at room temperature. The eluted phage were transfectedinto E. coli cells and allowed to grow overnight at 37° C. to amplifythe phage.

The second and third cycles of binding selection were carried out asabove, except that streptavidin (0.1 mg/mL) was included in the phagecocktails along with biotin. An aliquot was taken from eachtarget-coated and control well incubated with each library, and serialdilutions of the diluted phage were performed to measure specificbinding to target. The diluted phage were then transfected into E. colicells and plated for colony counting.

The fourth round of binding selection was carried out on MAXISORP™plates directly coated with 2 μg/ml of each target protein, or withalbumin only. The results of phage-binding selections in cycles 2-4 areshown in FIG. 25.

The same initial phage libraries (A, B, C, D) were also used for bindingselections to directly-coated IGFBP-3. In this case, MAXISORP™ 96-wellplastic plates (Nunc) were coated with a solution of 2 μg/ml of IGFBP-3in 50 mM sodium carbonate buffer, pH 9.6, overnight at 4° C. The targetsolution was then removed, and the plates were incubated with a blockingsolution of 5 g/L of bovine serum albumin, for 1-2 h at roomtemperature. Phage were incubated with the plates as above, andnon-binding phage washed away. The phage remaining bound were eluted byincubating with 20 mM HCl for 10 min at room temperature. Thereafter,the acid-eluted phage were neutralized with one-fifth volume of 1 MTris-HCl, pH 8.0. Phage were transfected for colony counting asdescribed above.

III. Screening of polyvalent phase clones (IGF-blocking phage assay)

Peptide-phage clones were isolated by mixing phage pools with E. colicells, and plating onto antibiotic-containing media.

Colonies were isolated and grown with helper phage (as above) to obtainsingle-stranded DNA for sequencing. Peptide sequences selected forbinding IGFBP-3, IGFBP-1, or IGF-I were deduced from the DNA sequencesof phagemid clones. A number of such clones are represented by thepeptide sequences in Tables III-V, respectively.

                  TABLE III                                                       ______________________________________                                        Peptide sequences from g8 display, IGFBP-3 selection                            Name      Peptide sequence                                                  ______________________________________                                        4A3.1   SGTACYGGPEWWCCSLAGSP                                                                              (SEQ ID NO: 59)                                     4A3.3 SGTACYGGPEWWCCSLAGSP (SEQ ID NO: 60)                                    4A3.4 SGTACYGGPEWWCCSLAGSP (SEQ ID NO: 61)                                    4B3.1 DLAICAEGPEIWVCEETS (SEQ ID NO: 62)                                      4B3.2 DFWICLSGPGWEECLEWW (SEQ ID NO: 63)                                      4B3.3 EESECFEGPGYVICGLVG (SEQ ID NO: 7)                                       4B3.4 DMGVCADGPWMYVCEWTE (SEQ ID NO: 8)                                       4B3.5 DMGVCADGPWMYVCEWTE (SEQ ID NO: 8)                                       4C3.1 GSAGQGMTEEWAWIWEWWKE (SEQ ID NO: 64)                                    4C3.2 ELDGWVCIKVGEQNLCYLAE (SEQ ID NO: 65)                                    4C3.4 ELDGWVCIKVGEQNLCYLAE (SEQ ID NO: 66)                                    4C3.4 ELDGWVCIKVGEQNLCYLAE (SEQ ID NO: 67)                                    4C3.5 ELDGWVCIKVGEQNLCYLAE (SEQ ID NO: 68)                                    4D3.1 AIGGWCFIELDSLWCEEQIG (SEQ ID NO: 69)                                    4D3.2 SEDVECWQVWENLVCSVEHR (SEQ ID NO: 70)                                    4D3.3 SEEVCWPVAEWYLCNMWGR (SEQ ID NO: 71)                                     4D3.4 RVGAYISCSETECWVEDLLD (SEQ ID NO: 72)                                    4D3.5 WFKTVCYEWEDEVQCYTLEE (SEQ ID NO: 73)                                    4D3.6 SEDVECWQVWENLVCSVEHR (SEQ ID NO: 74)                                    4D3.7 RLEEQCVEVNYEPSCSFTAN (SEQ ID NO: 75)                                    4D3.8 SEEVCWPVAEWYLCNILGP (SEQ ID NO: 76)                                     4D3.9 ETVANCDCYMDLCLCYGSDR (SEQ ID NO: 77)                                    4D3.10 YHPISCMDHYYLIICDETVN (SEQ ID NO: 78)                                   4D3.11 VAWEVCWDRHDQGYICTTDS (SEQ ID NO: 4)                                    4D3.12 AEWAECWIAGDQLLCVGKDN (SEQ ID NO: 79)                                   23A3.1 EPWLCQYYEAAMLYLCWEEG (SEQ ID NO: 80)                                   23A3.2 AEEGMVWGWTGGWYNLDELC (SEQ ID NO: 81)                                   23A3.3 SGGAIYWPVEQFIAFMAVGK (SEQ ID NO: 82)                                   23A3.4 EPWLCQYYEAAMLYLCWEEG (SEQ ID NO: 83)                                   23A3.5 SGGAIYMPVEQFIAFMAVGK (SEQ ID NO: 84)                                   23B3.1 TGVDCQCGPVHCVCMDWA (SEQ ID NO: 13)                                     23B3.2 EVLLCSDGPQLYLCELYA (SEQ ID NO: 85)                                     23B3.4 SGVECVWGPQWGFCVEEY (SEQ ID NO: 86)                                     23B3.5 DKEVCYLGPETWLCFWWP (SEQ ID NO: 87)                                     23B3.6 EVLLCSDGPQLYLCELYA (SEQ ID NO: 88)                                     23B3.7 GDVECIEGPWGELCVWAD (SEQ ID NO: 89)                                     23D3.1 FGGWSCQPTWVDVYVCNFEE (SEQ ID NO: 90)                                   23D3.2 AMWVCVSDWETVEECIQYMY (SEQ ID NO: 91)                                   23D3.3 AMWVCVSDWETVEECIQYMY (SEQ ID NO: 92)                                   23D3.4 AMWVCVSDWETVEECIQYMY (SEQ ID NO: 93)                                   23D3.5 AMWVCVSDWETVEECIQYMY (SEQ ID NO: 94)                                   23D3.6 TNWFFVCESGHQDICWLAEE (SEQ ID NO: 95)                                 ______________________________________                                    

                  TABLE IV                                                        ______________________________________                                        Peptide sequences from g8 display, IGFBP-1 selection                            Clone   Peptide sequence     Library                                                                              Frequency                               ______________________________________                                        HL-1  SEVGCRAGPLQLCEKYF    A        6/6                                          (SEQ ID NO: 96)                                                              HL-14 SEVGCRAGPLQLCEKYF B 5/6                                                  (SEQ ID NO: 97)                                                              HL-13 KDPVCGEGPLMRICERLFG B 1/6                                                (SEQ ID NO: 98)                                                              HL-31 EVDGRWWIVETFLAKWDHMAG C 6/6                                              (SEQ ID NO: 99)                                                            ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Peptide sequences from g8 display, IGF-I selection                              Clone   Peptide sequence     Library                                                                              Frequency                               ______________________________________                                        HL-8  WVMECGAGPWPEGCTFML   B        5/6                                          (SEQ ID NO: 100)                                                             HL-26 RKTSQGRGQEMCWETGGCS C 1/6                                                (SEQ ID NO: 101)                                                             HL-25 SWERGELTYMKLCEYMRLQQ C 4/6                                               (SEQ ID NO: 102)                                                             HL-30 EHGRANCLITPEAGKLARVT C 1/6                                               (SEQ ID NO: 103)                                                           ______________________________________                                    

Such peptide-phage clones could represent specific target-bindingpeptides which either do or do not block ligand (IGF-I to IGFBP)binding, or any of a number of non-binding or background members of theselected pool. To distinguish among these possibilities, phage cloneswere tested for the ability to bind to IGFBP-1 or IGFBP-3 in thepresence and absence of IGF-I.

IGFBP-1 or IGFBP-3 was coated directly onto MAXISORP™ plates as above.Phage from clonal cultures were mixed with IGF-I (100 nM finalconcentration), and incubated with the immobilized IGFBP for 1 hour atroom temperature. The plates were then washed ten times, as above, and asolution of rabbit anti-phage antibody mixed with a goat-anti-rabbitconjugate of horseradish peroxidase was added. After an incubation of 1hour at room temperature, the plates were developed with a chromogenicsubstrate, o-phenylenediamine (Sigma). The reaction was stopped withaddition of 1/2 volume of 2.5 M H₂ SO₄. Optical density at 490 nm wasmeasured on a spectrophotometric plate reader.

Titration of several IGFBP-3-selected peptide-phage clones showed allwere inhibited by IGF-I for binding to IGFBP-3 at some phageconcentration (FIGS. 26 and 27). These peptides are thus likely tooccupy an overlapping site with the IGF-binding epitope on IGFBP-3.Titration of an IGFBP-1-selected peptide-phage clone HL-1 showed it wasinhibited by IGF-I for binding to IGFBP-1 at some phage concentration(FIG. 28). Additional peptide-phage clones were screened similarly, at alow concentration of phage, with and without IGF-I.

FIG. 29 shows the results of a blocking assay of several phagemid clonesderived from three rounds of DTT elution, followed by one round of HClelution, as described above. In each case, the phagemid clone was grownfrom a single colony overnight at 37° C. in a culture volume of 5 ml.The phage particles were precipitated and resuspended in 0.5 ml of PBSbuffer. A 50-fold dilution of each phage solution was made intoPBS/TWEEN™ buffer, and the phage were incubated with or without 100 nMIGF-I on an IGFBP-3-coated MAXISORP™ plate. As shown in FIG. 29, mostclones were >40% inhibited for binding to IGFBP-3 at these phageconcentrations, although clone 4D3.11 was only 5% inhibited under theseconditions.

FIG. 30 shows the results of a blocking assay of several phagemid clonesderived from three rounds of HCl elution, as described above. In eachcase, the phagemid clone was grown from a single colony overnight at 37°C. in a culture volume of 5 ml.

The phage particles were prepared as described above. In this case, asshown in FIG. 30, most clones were >80% inhibited for binding to IGFBP-3at these phage concentrations, although clones 23A3.3 and 23A3.5 wereonly about 20% inhibited under these conditions.

The variation in the degree to which phage binding is blocked by aconstant concentration of IGF-I, as a function of phage dilution (FIGS.26, 28), or as a function of peptide displayed (FIGS. 29-30), is ofinterest because, without being limited to any one theory, it may bepredictive of (1) the degree of overlap between IGF-I- andpeptide-binding epitopes on the IGFBP-3 molecule, and/or (2) therelative affinity of IGF-I versus phage-displayed peptide for binding toIGFBP-3. Since all peptide-phage clones tested here showed some degreeof inhibition with IGF-I, it is likely that the epitope for peptidebinding on IGFBP-3 for each lies within an area occupied by bound IGF-I.Peptide assays (see below) support this conclusion (i.e., case 1). Onthe other hand, without being limited to any one theory, it is possiblethat some peptide epitopes could be simply within an area for whichbinding of the phage particle displaying such peptides is stericallyexcluded by bound IGF-I.

The dependence of inhibition upon phage concentration, and thedifferences among phage clones (FIG. 26) may reflect case 2. Inparticular, phage clones whose binding to an IGFBP-3-coated plate wasinhibited only at low phage concentrations (e.g., 4D3.3, 4B3.4,corresponding to peptides BP3-01-ox and BP3-02-ox, respectively) appearto yield higher-affinity peptides (see below) for IGFBP-3 than do thosephage clones whose binding to an IGFBP-3-coated plate was inhibited bothat high and at low phage concentrations (e.g., 4C3.2, 4D3.5,corresponding to peptides BP3-23 and BP3-24, respectively).

Thus, this type of phage-titration blocking assay may be generallyuseful as a means to predict the relative affinities and inhibitorypotencies of peptides derived from phage-displayed libraries.

IV. Monovalent (g3) display of IGFBP-3-binding peptides

Affinity maturation of a peptide or protein sequence by successiverounds of random mutagenesis, selection, and propagation can beefficiently accomplished when the copy number of displayed peptides orproteins is limited. Bass et al., Proteins, 8: 309 (1990). Such anaffinity maturation process is illustrated by the affinity maturation ofhGH. U.S. Pat. No. 5,534,617. In this case, the copy number of displayedhGH was limited by fusing the displayed protein to g3, rather than to g8of bacteriophage particles, restricting the expression level of hGH, andusing a helper phage to supply wild-type g3p for phagemid packaging andpropagation.

To select for higher-affinity peptide variants from pools ofphage-displaying peptides on g8p, peptide cDNAs from two round-4 g8library pools, 4B and 4D, were transferred to a g3 vector for monovalentphage display. Binding selections were carried out for three rounds, asdescribed above, with acid elution of binding phage.

Peptide sequences obtained after three rounds of selections are shown inTable VI. Two clones, 4B3.3 and 4D3.11, dominated the selected pools,and were seen in the earlier, g8 phage selections. A third clone, 3Ai.2,represents a new peptide sequence that was not identified from g8display. In phage-ELISA competition assays, the apparent affinity of theg3-4B3.3 and g3-4D3.11 clones was <100 nM; however, the correspondingpeptides showed much weaker inhibition (see below).

                  TABLE VI                                                        ______________________________________                                        Peptide sequences from g3 display, IGFBP-3 selection                            Clone      Peptide sequence   Library                                                                             Frequency                               ______________________________________                                        3Ai.1 = 4B3.3                                                                          EESECFEGPGYVICGLVG 4B      6/10                                         (SEQ ID NO: 7)                                                               3Ai.2 VEDECWMGPDWAVCWTWG 4B 4/10                                               (SEQ ID NO: 104)                                                             3Bi.1 = 4D3.11 VAWEVCWDRHDQGYICTTDS 4D 10/10                                   (SEQ ID NO: 4)                                                             ______________________________________                                    

It is anticipated that affinity improvements can be obtained byiteratively mutating, selecting, and propagating peptide-phagelibraries, as described for hGH. See, e.g., U.S. Pat. No. 5,534,617.

V. Peptide assays

Peptides were synthesized corresponding to a number of phage-derivedsequences. In cases where two Cys residues were found in the peptidesequence, the disulfide (oxidized or "ox" suffix) monomeric form of thepeptide was prepared and purified. In cases where four Cys residues werefound, the {1-4, 2-3}disulfide form was prepared and purified.

The ability of these peptides to bind IGFBP-1 or IGFBP-3 and block IGF-Ibinding was tested in one or more of the following assays.

A. BIAcore™ competition assay (for IGFBP-3 binders)

IGF-I was immobilized on a dextran chip for inhibition assays using aBIAcore™ 2000 surface-plasmon-resonance device (BIAcore, Inc.,Piscataway, N.J.) to measure free binding protein. IGF-I wasbiotinylated as described above, and injected over a chip to whichstreptavidin had been coupled (BIAcore, Inc.) to give 400 to 800 RU(response units) of immobilized IGF-I. The IGF-I showed no detectabledissociation over the time course of each experiment. Serial dilutionsof peptide were mixed with a constant concentration (40 nM) of IGFBP-3.After incubation for >1 hour at room temperature, an aliquot of 20 μLwas injected at a flow rate of 20 μL/min over the IGF-I chip. Followingthe injection, a response reading was taken to measure the relativeamount of IGFBP-3 bound to the IGF-I.

The results (FIGS. 31-32) show a dose-response curve for each peptide'sinhibition of IGFBP-3 binding to the chip. In particular, the mosteffective inhibitors of IGFBP-3 binding tested were peptides BP3-01-ox(corresponding to phage clone 4D3.3), and a truncated form of thispeptide, BP15 (see Table VII). In that table, a disulfide bond is formedbetween the two Cys residues of each 2-Cys-containing peptide. Forpeptides containing four cysteines, the two Cys* residues form adisulfide bond and the remaining two form a second disulfide bond. Thesepeptides showed IC50's of 2 μM and 0.75 μM, respectively. Other peptidessuch as BP3-4D3.11 (phage clone 4D3.11 from g8 display and 3Bi.1 from g3display) showed inhibition with IC50's of <10 μM.

IGFBP-1 did not show binding to IGF-I immobilized in this manner.

                  TABLE VII                                                       ______________________________________                                        Inhibition of IGF-I binding to IGFBP-3 by synthetic peptides                                                     BIAcore ™                                   Assay IC50                                                                  Peptide Sequence (μM)                                                    ______________________________________                                        BP3-23    ELDGWVCIKVGEQNLCYLAEG-nh2                                                                          220                                               (SEQ ID NO: 1)                                                               BP3-24 WFKTVCYEWEDEVQCYTLEEG-nh2 100-300                                       (SEQ ID NO: 2)                                                               BP3-25 RVGAYISCSETECWVEDLLDG-nh2 >1000                                         (SEQ ID NO: 3)                                                               BP3-4D3.11 VAWEVCWDRHDQGYICTTDS <10                                            (SEQ ID NO: 4)                                                               BP3-4D3.11DEL AWEVCWDRHQGYICTTDS 80                                            (SEQ ID NO: 5)                                                               BP13 CWDRHDQGYICTTDS >1000                                                     (SEQ ID NO: 6)                                                               BP3-4B3.3 EESECFEGPGYVICGLVG 80                                                (SEQ ID NO: 7)                                                               BP3-02-OX DMGVCADGPWMYVCEWTE 12                                                (SEQ ID NO: 8)                                                               BP3-01-OX SEEVCWPVAEWYLCNMWG 2                                                 (SEQ ID NO: 9)                                                               BP15 SEEVCWPVAEWYLCN 0.75                                                      (SEQ ID NO: 10)                                                              BP16 VCWPVAEWYLCNMWG 30                                                        (SEQ ID NO: 11)                                                              BP17 VCWPVAEWYLCN 9                                                            (SEQ ID NO: 12)                                                              BP06 TGVDCQC*GPVHC*VCMDWA 5                                                    (SEQ ID NO: 13)                                                              BP08 TVANCDC*YMPLC*LCYDSD 15                                                   (SEQ ID NO: 14)                                                            ______________________________________                                    

where nh2 means that the peptide has been blocked with an amide andwhere the C* indicates a cysteine that has been linked to anothercysteine in the peptide.

B. Plate assays

1. Biotin-BP assay (for IGFBP-1 binders)

For inhibition of IGFBP-1 binding, IGFBP-1 (as well as IGFBP-3 as acontrol) were biotinylated as described above. MAXISORP™ plates werecoated and blocked as described above, using 2 μg/mL IGF-I. In aseparate plate, serial dilutions of peptide were premixed with aconstant concentration (20 nM) of biotinylated IGFBP-1. After 20 min. atroom temperature, the mixture was added to the IGF-I plate. Thepeptide/IGFBP mix was then removed and the plate was washed ten timeswith PBS buffer containing 0.05% TWEEN-20™ surfactant. Boundbiotin-IGFBP-1 was then detected using streptavidin-conjugatedhorseradish peroxidase, and a chromogenic substrate.

The results of assays of IGFBP-1 inhibition (FIGS. 33 and 34) show thatpeptides bp1-01 and bp1-02 (Table VIII) inhibited IGFBP-1 binding withIC50's of about 0.18 and 0.05 μM, respectively. In contrast, the IGFBP-3selectant BP3-01-ox showed little or no inhibition of IGFBP-1 binding.Conversely, bp1-01 and bp1-02 showed no inhibition of biotinylatedIGFBP-3 binding in this assay. A bp1-01 variant in which the Cysresidues were changed to serine showed an IC50 of greater than 10 μM.

                  TABLE VIII                                                      ______________________________________                                        Inhibition of IGF-I binding to IGFBP-1 by synthetic peptides                    Peptide  Sequence             ELISA IC50 (μM)                            ______________________________________                                        bp1-01 CRAGPLQWLCEKYFG-nh2  0.18                                                 (SEQ ID NO: 15)                                                              bp1-02 SEVGCRAGPLQWLCEKYFG-nh2 0.05                                            (SEQ ID NO: 16)                                                            ______________________________________                                    

2. Radiolabeled IGF assay (for IGFBP-3 binders)

As an additional assay of peptide activity, several peptides were testedin an assay using ¹²⁵ I-labeled IGF-I to measure inhibition of IGFBPbinding, as described in Example 1 (Assay 3). Serial dilutions ofpeptide were added to an IGFBP-1 or an IGFBP-3 plate. Thereafter, ¹²⁵I-labeled IGF-I was added and the plates were incubated for 2 hours. Theplates were then washed and counted to determine the amount of boundIGF-I.

FIG. 35 shows the inhibition of two IGFBP-3-selected peptides, bp3-01-oxand bp3-02-ox, for IGF-I binding to an IGFBP-3 plate. In contrast, thesepeptides did not inhibit IGF-I binding to an IGFBP-1-coated plate (FIG.36).

C. In vitro activation (KIRA)

The ability of several synthetic peptides to block IGF-I binding toIGFBPs and release functional IGF-I was tested in a KIRA assay of IGF-Iactivity, as described in Example 1. Cells were treated with IGF-Ialone, peptide alone, peptide plus IGF-I, IGF-I plus binding protein(IGFBP-1 or IGFBP-3), or IGF-I plus binding protein (IGFBP-1 or IGFBP-3)and peptide.

The results (FIG. 37) show that the peptides alone had no activity.Furthermore, when mixed with IGF-I, the peptides did not significantlyalter IGF-I activity. The peptide BP3-01-ox also showed no significanteffect on IGF-I activity when mixed with IGF-I plus IGFBP-l or IGFBP-3.However, both peptides bp1-01 and bp1-02 appeared to block theinhibition of IGF-I activity by IGFBP-1, with IC50's of 397 nM and 187nM, respectively. Neither of these peptides showed activity with IGF-Iand IGFBP-3.

In addition, the IGFBP-3 peptide BP15 incubated with IGFBP-3:IGF-1 (at a3:1 ratio) caused an activation of the KIRA assay, where blocking ofIGFBP-3 occurred with an IC50 of 95 μM for BP15. It is believed thatthis would drop to 30 μM with a 1:1 ratio.

D. BIAcore™ competition assay (for IGFBP-3 binders)

IGF-II was immobilized on a dextran chip for inhibition assays using aBIAcore™ 2000 surface-plasmon-resonance device (BIAcore, Inc.,Piscataway, N.J.) to measure free binding protein. IGF-II wasbiotinylated as described above, and injected over a chip to whichstreptavidin had been coupled (BIAcore, Inc.) to give approximately 1500RU of immobilized IGF-II. The IGF-II showed no detectable dissociationover the time course of each experiment. Serial dilutions of peptidewere mixed with a constant concentration (20 nM) of IGFBP-3. Afterincubation for ≧1 hour at room temperature, an aliquot of 20 μl wasinjected at a flow rate of 20 μl/min over the IGF-II chip. Following theinjection, a response reading was taken to measure the relative amountof IGFBP-3 bound to the IGF-II.

The results (e.g., see FIG. 38) show a dose-response curve for eachpeptide's inhibition of IGFBP-3 binding to IGF-II. Peptides bp3-01-ox,BP14, BP15, and BP17 showed IC50's of 0.92 μM, 1.0 μM, 0.78 μM, and 5.1μM, respectively. Thus, these peptides inhibit the binding of IGFBP-3both to IGF-I and to IGF-II.

Example 8 NMR Spectra of Peptide bp1-01

¹ H NMR data were collected on peptide bp1-01 in H₂ O solution at 30° C.and pH 5.2 at a concentration of 6.7 millimolar. For generation of theone-dimensional NMR spectrum of FIG. 39, a total of 5.0 mg of purifiedpeptide was dissolved in 440 μl H₂ O containing 7% (v/v) ² H₂ O for thespectrometers field frequency lock. The pH of the sample was adjusted to5.2 by the addition of 3 μl of 1N NaOH. The spectrum consisting of 32transients was collected on a BRUKER AMX-500™ spectrometer (¹ Hfrequency of 500.13 MHz) equipped with a 5-mm triple-axis, pulsed-fieldgradient probe. The chemical shift scale is in parts per million,referenced to the water resonance at 4.75 p.p.m. The water resonance wassuppressed by the excitation sculpting method. Hwang and Shaka, J. Magn.Reson., 112A: 275-279 (1995).

In addition to the one-dimensional spectrum of FIG. 39, two-dimensionaldouble-quantum-filtered correlation spectroscopy (2QF-COSY), totalcorrelation spectra (TOCSY), and rotating-frame Overhauser effectspectra (ROESY) were collected. The experiments were recorded asdescribed by Cavanagh et al. in "Protein NMR Spectroscopy, Principlesand Practice" (Academic Press, San Diego: ISBN 0-12-164490-1, 1995),except that pulsed-field gradients were used for coherence selection inthe 2QF-COSY (van Zijl et al., J. Magn. Reson., 113A: 265-270 (1995)),and excitation sculpting was used to suppress the water resonance in theTOCSY and ROESY experiments (Hwang and Shaka, supra). Afterlyophilization and dissolution of the peptide in ² H₂ O, a 2-D ROESYspectrum (Cavanagh et al., supra) and a COSY spectrum with a 35° C.mixing pulse (Cavanagh et al., supra) were acquired. Complete ¹ Hresonance assignments were obtained from these data by standard methods.Wuethrich, in "NMR of proteins and nucleic acids", (John Wiley & Sons,New York: ISBN 0-471-82893-9, 1986).

Evidence of a well-defined three-dimensional structure for bp1-01 wasobtained from the following:

1) The resonance positions in the one-dimensional spectrum (FIG. 39) aresignificantly different from those expected in an unstructured peptide.For example, the amide protons span a range of 8.73-6.64 p.p.m.(unstructured peptides are in the range 8.50-8.00 p.p.m.); and themethyl groups are at lower chemical shift (0.82 p.p.m. for Leu6, and0.66, 0.59 p.p.m. for Leu9) than for an unstructured molecule (0.90-0.85p.p.m.).

2) Scalar coupling constants between amide and alpha protons (obtainedfrom the 2QF-COSY spectrum) are distinct from the averaged valuesobserved in unstructured peptides. The values less than 5.5 Hz for Gln7,Trp8, Cys10, Glu11, and Lys12 are indicative of a helix spanning theseresidues. The values greater than 8.0 Hz observed for Leu6, Tyr13, andPhe14 are indicative of an extended conformation in these regions.Scalar coupling constants were also measured between alpha and betaprotons in the COSY-35 spectrum. These data indicate that the sidechains of Cys1, Gln7, Trp8, Cys10, Tyr13, and Phe14 have fixed chi-1angles, i.e., these side chains do not sample the range of chi-1rotamers that are populated in unstructured peptides.

3) Peaks in the ROESY spectra indicate that there are manyproton--proton contacts (<5 Å) between residues that are not adjacent inthe primary sequence. These can only occur if the peptide folds up intoa well-defined structure. Contacts between residues at position i andi+3 in the primary sequence are prevalent between Leu6 and Tyr13,consistent with the presence of a helix in this region. Many contactsare observed between the three aromatic side chains (Trp8, Tyr13, andPhe14) and the leucine methyl groups (Leu6 and Leu9), indicating thepresence of a mini-hydrophobic core along one face of the helix.

The NMR data were used to derive restraints that could be used todetermine a three-dimensional model of the bp1-01 structure. Dihedralangle restraints were derived from the amide-alpha and alpha-beta scalarcoupling constants via an appropriate Karplus relationship. Karplus, J.Phys. Chem., 30: 11-15 (1959). Distance restraints were introducedbetween protons which exhibited a through-space interaction in the ROESYspectrum; the size of the upper bound, and corrections to the upperbound because of peak overlap or resonance degeneracy were as describedby Skelton et al., Biochemistry, 33: 13581-92 (1994). These restraintswere used to generate a family of structures using the program DGII(Havel, Prog. Biophys. Mol. Biol., 56: 43-78 (1991)), which weresubsequently refined by restrained molecular dynamics with the programDiscover (MSI, San Diego) using the AMBER all atom force field. Weineret al., J. Comput. Chem., 7: 230-252 (1986). The resulting structuresconverged to a single global fold (mean root-mean-squared deviation fromthe mean structure of 0.37 Å for N, C-alpha, and carbonyl carbons ofresidues 4-14). The best 25 structures (least violation of the inputrestraints) agreed with the input data very well (no distance restraintviolations greater than 0.1 Å and no dihedral angle violations greaterthan 1.4°), and had good covalent geometry as judged by the programPROCHECK™. Laskowski et al., J. Appl. Cryst., 26: 283-291 (1993).

A representative member of the ensemble is shown in FIGS. 40A and 40B.According to the Kabsch and Sander secondary structure algorithm withinthe PROCHECK™ program, bp1-01 contains a reverse turn centered atPro5-Leu6 (Type I) and an alpha helix from Gln7 to Lys12; Leu6 and Tyr13are extensions of the main helix. The residues Cys1, Arg2, Ala3, andGly15 are not well defined by the NMR restraints and may be moreflexible in solution than residues Gly4 to Phe14. FIGS. 40A and 40Bindicate that the aromatic rings of Trp8, Tyr13, and Phe13 pack onto thealiphatic portions of Leu6, Leu9, and Lys12 to form a relatively flat,hydrophobic face on one side of the peptide. This packing isaccomplished by the periodicity of the helix (bringing Leu6, Leu9, andTyr13 together), the chi-l angle of Trp8 (the chi-1 of +60 degrees,relatively unusual for this amino acid, allows Trp8 to pack onto Leu9),and the extended nature of the peptide backbone of Phe14 (allowing theside chain of Phe14 to fold back on to Leu9).

Although bp1-01 is prone to self-association under some conditions, thethree-dimensional structure observed for bp1-01 is not stabilized bysuch intermolecular interactions. The hydrophobic packing within thehelix undoubtedly contributes to the stability of the fold. Thedisulfide bond between Cys1 and Cys10 is also important for structureand function. Peptides in which both cystine residues are replaced byserine have much reduced affinity for IGFBP-1 (see Example 7) andexhibit no evidence of helical or other stable structure in one- andtwo-dimensional NMR spectra, suggesting that the structure displayed bybp1-01 is important for its function. Without being limited to any onetheory, one possibility is that the reverse turn (Pro5-Leu6) anddisulfide bond help to initiate the first turn of helix (Gln7-Cys10),which is then propagated by the hydrophobic side chain-side chaincontacts involving residues in the second turn of helix (Glu11-Phe14).

The structure of bp1-01 shown in FIG. 40 suggests several possibilitiesfor the way in which the peptide is able to bind to IGFBP-l, therebyenhancing levels of active IGF-I.

1) The flat, hydrophobic surface formed by the side chains of Leu6,Leu9, Trp8, Tyr13, and Phe14 in the bp1-01 monomer may pack onto anexposed hydrophobic region of IGFBP-1; in this case the helix is the keystructural feature recognized by IGFBP-1;

2) BP-1 may recognize some part of bp1-01 other than the hydrophobicface of the helix, e.g., the five N-terminal residues preceding thehelix, or a combination of these residues and the face of the helix awayfrom hydrophobic side chains. In this case, the helix may still beimportant as a scaffold to set up a geometry appropriate for IGFBP-1binding elsewhere in the peptide.

3) bp1-01 may bind to IGFBP-1 in an aggregated form. Given thelikelihood of self-association of bp1-01 via the hydrophobic face of thehelix (Leu6, Trp8, Leu9, Tyr13, and Phe14), some other region of thepeptide would be involved in IGFBP-1 contact. In this case the helicesand the contacts between them would act as a scaffold to present theIGFBP-l binding region.

Below are the coordinates for the representative structure that is shownin FIG. 39 (standard protein database format).

    __________________________________________________________________________    REMARK BP1-01 IGF-I-BINDING PROTEIN BINDING PEPTIDE                             REMARK REPRESENTATIVE STRUCTURE FROM ENSEMBLE CALCULATED                      REMARK WITH NMR-DERIVED RESTRAINTS                                          ATOM  1   N   CYS 1  -5.022                                                                             -1.559                                                                             0.892                                                                              1.00                                                                              0.00                                    ATOM  2  CA CYS 1 -4.328 -1.133 -0.335 1.00 0.00                              ATOM  3  C CYS 1 -3.837 0.310 -0.220 1.00 0.00                                ATOM  4  O CYS 1 -2.646 0.544 -0.024 1.00 0.00                                ATOM  5  CB CYS 1 -5.220 -1.340 -1.567 1.00 0.00                              ATOM  6  SG CYS 1 -4.498 -0.877 -3.166 1.00 0.00                              ATOM  7 1H CYS 1 -5.842 -0.987 1.034 1.00 0.00                                ATOM  8 2H CYS 1 -5.296 -2.527 0.807 1.00 0.00                                ATOM  9 3H CYS 1 -4.400 -1.455 1.682 1.00 0.00                                ATOM  10  HA CYS 1 -3.456 -1.777 -0.448 1.00 0.00                             ATOM  11 1HB CYS 1 -5.484 -2.396 -1.625 1.00 0.00                             ATOM  12 2HB CYS 1 -6.143 -0.773 -1.446 1.00 0.00                             ATOM  13 1LG CYS 1 -4.970 -1.181 -3.550 1.00 0.00                             ATOM  14 2LG CYS 1 -4.093 -0.385 -2.924 1.00 0.00                             ATOM  15  N ARG 2 -4.756 1.275 -0.336 1.00 0.00                               ATOM  16  CA ARG 2 -4.410 2.685 -0.348 1.00 0.00                              ATOM  17  C ARG 2 -3.778 3.012 -1.703 1.00 0.00                               ATOM  18  O ARG 2 -2.559 3.112 -1.836 1.00 0.00                               ATOM  19  CB ARG 2 -3.519 2.999 0.862 1.00 0.00                               ATOM  20  CG ARG 2 -3.052 4.460 0.950 1.00 0.00                               ATOM  21  CD ARG 2 -4.211 5.459 0.892 1.00 0.00                               ATOM  22  NE ARG 2 -3.742 6.823 1.164 1.00 0.00                               ATOM  23  CZ ARG 2 -4.455 7.940 0.945 1.00 0.00                               ATOM  24  NH1 ARG 2 -5.701 7.877 0.455 1.00 0.00                              ATOM  25  NH2 ARG 2 -3.914 9.134 1.222 1.00 0.00                              ATOM  26  H ARG 2 -5.723 1.024 -0.475 1.00 0.00                               ATOM  27  HA ARG 2 -5.340 3.246 -0.248 1.00 0.00                              ATOM  28 1HB ARG 2 -4.084 2.770 1.767 1.00 0.00                               ATOM  29 2HB ARG 2 -2.647 2.346 0.839 1.00 0.00                               ATOM  30 1HG ARG 2 -2.523 4.585 1.896 1.00 0.00                               ATOM  31 2HG ARG 2 -2.356 4.680 0.142 1.00 0.00                               ATOM  32 1HD ARG 2 -4.657 5.430 -0.103 1.00 0.00                              ATOM  33 2HD ARG 2 -4.962 5.189 1.635 1.00 0.00                               ATOM  34  HE ARG 2 -2.813 6.913 1.550 1.00 0.00                               ATOM  35 2HH1 ARG 2 -6.227 8.724 0.294 1.00 0.00                              ATOM  36 1HH1 ARG 2 -6.122 6.981 0.254 1.00 0.00                              ATOM  37 1HH2 ARG 2 -2.979 9.192 1.599 1.00 0.00                              ATOM  38 2HH2 ARG 2 -4.442 9.979 1.061 1.00 0.00                              ATOM  39  N ALA 3 -4.640 3.150 -2.718 1.00 0.00                               ATOM  40  CA ALA 3 -4.259 3.352 -4.106 1.00 0.00                              ATOM  41  C ALA 3 -3.843 4.805 -4.352 1.00 0.00                               ATOM  42  O ALA 3 -4.546 5.555 -5.027 1.00 0.00                               ATOM  43  CB ALA 3 -5.430 2.931 -5.001 1.00 0.00                              ATOM  44  H ALA 3 -5.626 3.058 -2.522 1.00 0.00                               ATOM  45  HA ALA 3 -3.412 2.706 -4.345 1.00 0.00                              ATOM  46 3HB ALA 3 -5.643 1.872 -4.851 1.00 0.00                              ATOM  47 1HB ALA 3 -6.320 3.511 -4.753 1.00 0.00                              ATOM  48 2HB ALA 3 -5.182 3.096 -6.049 1.00 0.00                              ATOM  49  N GLY 4 -2.681 5.191 -3.818 1.00 0.00                               ATOM  50  CA GLY 4 -2.069 6.481 -4.087 1.00 0.00                              ATOM  51  C GLY 4 -0.636 6.488 -3.558 1.00 0.00                               ATOM  52  O GLY 4 0.272 6.072 -4.276 1.00 0.00                                ATOM  53  H GLY 4 -2.169 4.528 -3.249 1.00 0.00                               ATOM  54 1HA GLY 4 -2.035 6.637 -5.166 1.00 0.00                              ATOM  55 2HA GLY 4 -2.666 7.280 -3.648 1.00 0.00                              ATOM  56  N PRO 5 -0.410 6.939 -2.314 1.00 0.00                               ATOM  57  CA PRO 5 0.917 7.007 -1.721 1.00 0.00                               ATOM  58  C PRO 5 1.505 5.610 -1.509 1.00 0.00                                ATOM  59  O PRO 5 2.698 5.413 -1.731 1.00 0.00                                ATOM  60  CB PRO 5 0.739 7.773 -0.407 1.00 0.00                               ATOM  61  CG PRO 5 -0.715 7.501 -0.026 1.00 0.00                              ATOM  62  CD PRO 5 -1.419 7.411 -1.380 1.00 0.00                              ATOM  63  HA PRO 5 1.586 7.574 -2.370 1.00 0.00                               ATOM  64 1HB PRO 5 1.439 7.458 0.368 1.00 0.00                                ATOM  65 2HB PRO 5 0.857 8.840 -0.600 1.00 0.00                               ATOM  66 1HG PRO 5 -0.781 6.542 0.486 1.00 0.00                               ATOM  67 2HG PRO 5 -1.126 8.290 0.603 1.00 0.00                               ATOM  68 1HD PRO 5 -2.270 6.732 -1.322 1.00 0.00                              ATOM  69 2HD PRO 5 -1.752 8.405 -1.683 1.00 0.00                              ATOM  70  N LEU 6 0.670 4.646 -1.098 1.00 0.00                                ATOM  71  CA LEU 6 1.043 3.246 -0.926 1.00 0.00                               ATOM  72  C LEU 6 0.520 2.418 -2.110 1.00 0.00                                ATOM  73  O LEU 6 0.206 1.241 -1.944 1.00 0.00                                ATOM  74  CB LEU 6 0.497 2.733 0.422 1.00 0.00                                ATOM  75  CG LEU 6 1.343 3.106 1.651 1.00 0.00                                ATOM  76  CD1 LEU 6 1.406 4.613 1.920 1.00 0.00                               ATOM  77  CD2 LEU 6 0.748 2.411 2.883 1.00 0.00                               ATOM  78  H LEU 6 -0.298 4.883 -0.937 1.00 0.00                               ATOM  79  HA LEU 6 2.127 3.131 -0.913 1.00 0.00                               ATOM  80 1HB LEU 6 -0.520 3.095 0.556 1.00 0.00                               ATOM  81 2HB LEU 6 0.465 1.645 0.409 1.00 0.00                                ATOM  82  HG LEU 6 2.359 2.738 1.507 1.00 0.00                                ATOM  83 2HD1 LEU 6 1.891 4.795 2.879 1.00 0.00                               ATOM  84 3HD1 LEU 6 1.994 5.109 1.150 1.00 0.00                               ATOM  85 1HD1 LEU 6 0.400 5.032 1.947 1.00 0.00                               ATOM  86 2HD2 LEU 6 1.368 2.612 3.757 1.00 0.00                               ATOM  87 3HD2 LEU 6 -0.261 2.780 3.069 1.00 0.00                              ATOM  88 1HD2 LEU 6 0.707 1.332 2.728 1.00 0.00                               ATOM  89  N GLN 7 0.441 3.001 -3.316 1.00 0.00                                ATOM  90  CA GLN 7 0.009 2.262 -4.497 1.00 0.00                               ATOM  91  C GLN 7 1.046 1.194 -4.848 1.00 0.00                                ATOM  92  O GLN 7 0.696 0.030 -5.022 1.00 0.00                                ATOM  93  CB GLN 7 -0.227 3.214 -5.677 1.00 0.00                              ATOM  94  CG GLN 7 -0.648 2.494 -6.968 1.00 0.00                              ATOM  95  CD GLN 7 -1.990 1.773 -6.841 1.00 0.00                              ATOM  96  OE1 GLN 7 -3.015 2.295 -7.270 1.00 0.00                             ATOM  97  NE2 GLN 7 -1.991 0.566 -6.271 1.00 0.00                             ATOM  98  H GLN 7 0.702 3.971 -3.428 1.00 0.00                                ATOM  99  HA GLN 7 -0.936 1.778 -4.245 1.00 0.00                              ATOM 100 1HB GLN 7 -1.007 3.925 -5.415 1.00 0.00                              ATOM 101 2HB GLN 7 0.690 3.769 -5.878 1.00 0.00                               ATOM 102 1HG GLN 7 -0.742 3.247 -7.752 1.00 0.00                              ATOM 103 2HG GLN 7 0.120 1.787 -7.282 1.00 0.00                               ATOM 104 1HE2 GLN 7 -2.855 0.049 -6.189 1.00 0.00                             ATOM 105 2HE2 GLN 7 -1.129 0.171 -5.919 1.00 0.00                             ATOM 106  N TRP 8 2.319 1.596 -4.940 1.00 0.00                                ATOM 107  CA TRP 8 3.449 0.700 -5.154 1.00 0.00                               ATOM 108  C TRP 8 3.434 -0.449 -4.141 1.00 0.00                               ATOM 109  O TRP 8 3.652 -1.602 -4.505 1.00 0.00                               ATOM 110  CB TRP 8 4.756 1.500 -5.048 1.00 0.00                               ATOM 111  CG TRP 8 5.026 2.107 -3.702 1.00 0.00                               ATOM 112  CD1 TRP 8 4.543 3.290 -3.263 1.00 0.00                              ATOM 113  CD2 TRP 8 5.753 1.533 -2.573 1.00 0.00                              ATOM 114  NE1 TRP 8 4.896 3.483 -1.944 1.00 0.00                              ATOM 115  CE2 TRP 8 5.643 2.425 -1.466 1.00 0.00                              ATOM 116  CE3 TRP 8 6.450 0.324 -2.354 1.00 0.00                              ATOM 117  CZ2 TRP 8 6.203 2.134 -0.213 1.00 0.00                              ATOM 118  CZ3 TRP 8 7.061 0.049 -1.117 1.00 0.00                              ATOM 119  CH2 TRP 8 6.933 0.948 -0.045 1.00 0.00                              ATOM 120  H TRP 8 2.521 2.575 -4.803 1.00 0.00                                ATOM 121  HA TRP 8 3.378 0.286 -6.161 1.00 0.00                               ATOM 122 1HB TRP 8 5.585 0.835 -5.293 1.00 0.00                               ATOM 123 2HB TRP 8 4.739 2.297 -5.793 1.00 0.00                               ATOM 124  HD1 TRP 8 3.930 3.966 -3.841 1.00 0.00                              ATOM 125  HE1 TRP 8 4.624 4.279 -1.384 1.00 0.00                              ATOM 126  HE3 TRP 8 6.504 -0.411 -3.142 1.00 0.00                             ATOM 127  HZ2 TRP 8 6.053 2.799 0.624 1.00 0.00                               ATOM 128  HZ3 TRP 8 7.623 -0.862 -0.986 1.00 0.00                             ATOM 129  HH2 TRP 8 7.382 0.719 0.911 1.00 0.00                               ATOM 130  N LEU 9 3.164 -0.117 -2.872 1.00 0.00                               ATOM 131  CA LEU 9 3.155 -1.044 -1.753 1.00 0.00                              ATOM 132  C LEU 9 2.024 -2.054 -1.956 1.00 0.00                               ATOM 133  O LEU 9 2.239 -3.260 -1.862 1.00 0.00                               ATOM 134  CB LEU 9 3.025 -0.233 -0.448 1.00 0.00                              ATOM 135  CG LEU 9 3.830 -0.813 0.726 1.00 0.00                               ATOM 136  CD1 LEU 9 3.898 0.197 1.877 1.00 0.00                               ATOM 137  CD2 LEU 9 3.197 -2.091 1.264 1.00 0.00                              ATOM 138  H LEU 9 2.975 0.853 -2.669 1.00 0.00                                ATOM 139  HA LEU 9 4.112 -1.567 -1.754 1.00 0.00                              ATOM 140 1HB LEU 9 3.418 0.768 -0.625 1.00 0.00                               ATOM 141 2HB LEU 9 1.977 -0.133 -0.166 1.00 0.00                              ATOM 142  HG LEU 9 4.846 -1.031 0.399 1.00 0.00                               ATOM 143 3HD1 LEU 9 4.523 -0.200 2.678 1.00 0.00                              ATOM 144 1HD1 LEU 9 4.326 1.137 1.535 1.00 0.00                               ATOM 145 2HD1 LEU 9 2.899 0.384 2.267 1.00 0.00                               ATOM 146 2HD2 LEU 9 3.808 -2.482 2.077 1.00 0.00                              ATOM 147 3HD2 LEU 9 2.197 -1.867 1.630 1.00 0.00                              ATOM 148 1HD2 LEU 9 3.135 -2.835 0.479 1.00 0.00                              ATOM 149  N CYS 10 0.830 -1.551 -2.288 1.00 0.00                              ATOM 150  CA CYS 10 -0.347 -2.351 -2.581 1.00 0.00                            ATOM 151  C CYS 10 -0.090 -3.354 -3.706 1.00 0.00                             ATOM 152  O CYS 10 -0.370 -4.536 -3.533 1.00 0.00                             ATOM 153  CB CYS 10 -1.528 -1.430 -2.894 1.00 0.00                            ATOM 154  SG CYS 10 -3.024 -2.270 -3.462 1.00 0.00                            ATOM 155  H CYS 10 0.733 -0.547 -2.354 1.00 0.00                              ATOM 156  HA CYS 10 -0.598 -2.913 -1.684 1.00 0.00                            ATOM 157 1HB CYS 10 -1.764 -0.865 -1.995 1.00 0.00                            ATOM 158 2HB CYS 10 -1.251 -0.720 -3.670 1.00 0.00                            ATOM 159 1LG CYS 10 -2.931 -2.146 -4.124 1.00 0.00                            ATOM 160 2LG CYS 10 -3.134 -2.620 -2.887 1.00 0.00                            ATOM 161  N GLU 11 0.449 -2.907 -4.846 1.00 0.00                              ATOM 162  CA GLU 11 0.729 -3.800 -5.964 1.00 0.00                             ATOM 163  C GLU 11 1.792 -4.838 -5.593 1.00 0.00                              ATOM 164  O GLU 11 1.609 -6.024 -5.859 1.00 0.00                              ATOM 165  CB GLU 11 1.168 -3.009 -7.206 1.00 0.00                             ATOM 166  CG GLU 11 0.102 -2.037 -7.727 1.00 0.00                             ATOM 167  CD GLU 11 -1.243 -2.716 -7.973 1.00 0.00                            ATOM 168  OE1 GLU 11 -1.333 -3.451 -8.980 1.00 0.00                           ATOM 169  OE2 GLU 11 -2.154 -2.493 -7.146 1.00 0.00                           ATOM 170  H GLU 11 0.669 -1.924 -4.942 1.00 0.00                              ATOM 171  HA GLU 11 -0.182 -4.348 -6.204 1.00 0.00                            ATOM 172 1HB GLU 11 2.075 -2.446 -6.979 1.00 0.00                             ATOM 173 2HB GLU 11 1.396 -3.721 -8.001 1.00 0.00                             ATOM 174 1HG GLU 11 -0.034 -1.231 -7.014 1.00 0.00                            ATOM 175 2HG GLU 11 0.452 -1.601 -8.663 1.00 0.00                             ATOM 176  N LYS 12 2.909 -4.388 -5.010 1.00 0.00                              ATOM 177  CA LYS 12 4.080 -5.221 -4.779 1.00 0.00                             ATOM 178  C LYS 12 3.824 -6.277 -3.701 1.00 0.00                              ATOM 179  O LYS 12 4.069 -7.459 -3.936 1.00 0.00                              ATOM 180  CB LYS 12 5.280 -4.325 -4.444 1.00 0.00                             ATOM 181  CG LYS 12 6.590 -5.123 -4.400 1.00 0.00                             ATOM 182  CD LYS 12 7.820 -4.206 -4.385 1.00 0.00                             ATOM 183  CE LYS 12 7.961 -3.450 -3.061 1.00 0.00                             ATOM 184  NZ LYS 12 9.120 -2.542 -3.084 1.00 0.00                             ATOM 185  H LYS 12 2.989 -3.401 -4.803 1.00 0.00                              ATOM 186  HA LYS 12 4.306 -5.736 -5.715 1.00 0.00                             ATOM 187 1HB LYS 12 5.366 -3.568 -5.225 1.00 0.00                             ATOM 188 2HB LYS 12 5.107 -3.827 -3.490 1.00 0.00                             ATOM 189 1HG LYS 12 6.604 -5.776 -3.526 1.00 0.00                             ATOM 190 2HG LYS 12 6.650 -5.743 -5.296 1.00 0.00                             ATOM 191 1HD LYS 12 8.711 -4.820 -4.529 1.00 0.00                             ATOM 192 2HD LYS 12 7.754 -3.496 -5.210 1.00 0.00                             ATOM 193 1HE LYS 12 7.068 -2.857 -2.869 1.00 0.00                             ATOM 194 2HE LYS 12 8.098 -4.170 -2.255 1.00 0.00                             ATOM 195 2HZ LYS 12 9.000 -1.861 -3.820 1.00 0.00                             ATOM 196 3HZ LYS 12 9.192 -2.066 -2.196 1.00 0.00                             ATOM 197 1HZ LYS 12 9.962 -3.073 -3.250 1.00 0.00                             ATOM 198  N TYR 13 3.345 -5.856 -2.524 1.00 0.00                              ATOM 199  CA TYR 13 3.118 -6.740 -1.388 1.00 0.00                             ATOM 200  C TYR 13 1.704 -7.312 -1.433 1.00 0.00                              ATOM 201  O TYR 13 1.537 -8.530 -1.445 1.00 0.00                              ATOM 202  CB TYR 13 3.347 -5.993 -0.069 1.00 0.00                             ATOM 203  CG TYR 13 4.787 -5.600 0.198 1.00 0.00                              ATOM 204  CD1 TYR 13 5.300 -4.391 -0.305 1.00 0.00                            ATOM 205  CD2 TYR 13 5.596 -6.412 1.016 1.00 0.00                             ATOM 206  CE1 TYR 13 6.582 -3.958 0.070 1.00 0.00                             ATOM 207  CE2 TYR 13 6.896 -6.000 1.354 1.00 0.00                             ATOM 208  CZ TYR 13 7.385 -4.766 0.892 1.00 0.00                              ATOM 209  OH TYR 13 8.639 -4.357 1.241 1.00 0.00                              ATOM 210  H TYR 13 3.140 -4.873 -2.402 1.00 0.00                              ATOM 211  HA TYR 13 3.826 -7.571 -1.417 1.00 0.00                             ATOM 212 1HB TYR 13 2.720 -5.102 -0.042 1.00 0.00                             ATOM 213 2HB TYR 13 3.018 -6.640 0.745 1.00 0.00                              ATOM 214  HD1 TYR 13 4.707 -3.781 -0.968 1.00 0.00                            ATOM 215  HD2 TYR 13 5.218 -7.350 1.396 1.00 0.00                             ATOM 216  HE1 TYR 13 6.948 -3.005 -0.278 1.00 0.00                            ATOM 217  HE2 TYR 13 7.514 -6.626 1.982 1.00 0.00                             ATOM 218  HH TYR 13 8.872 -3.497 0.883 1.00 0.00                              ATOM 219  N PHE 14 0.685 -6.443 -1.441 1.00 0.00                              ATOM 220  CA PHE 14 -0.707 -6.851 -1.269 1.00 0.00                            ATOM 221  C PHE 14 -1.342 -7.174 -2.628 1.00 0.00                             ATOM 222  O PHE 14 -2.470 -6.768 -2.906 1.00 0.00                             ATOM 223  CB PHE 14 -1.488 -5.752 -0.525 1.00 0.00                            ATOM 224  CG PHE 14 -0.808 -5.116 0.680 1.00 0.00                             ATOM 225  CD1 PHE 14 0.044 -5.862 1.519 1.00 0.00                             ATOM 226  CD2 PHE 14 -1.039 -3.756 0.964 1.00 0.00                            ATOM 227  CE1 PHE 14 0.722 -5.231 2.578 1.00 0.00                             ATOM 228  CE2 PHE 14 -0.370 -3.129 2.027 1.00 0.00                            ATOM 229  CZ PHE 14 0.523 -3.862 2.826 1.00 0.00                              ATOM 230  H PHE 14 0.882 -5.453 -1.475 1.00 0.00                              ATOM 231  HA PHE 14 -0.751 -7.755 -0.659 1.00 0.00                            ATOM 232 1HB PHE 14 -1.708 -4.956 -1.237 1.00 0.00                            ATOM 233 2HB PHE 14 -2.441 -6.169 -0.195 1.00 0.00                            ATOM 234  HD1 PHE 14 0.196 -6.918 1.352 1.00 0.00                             ATOM 235  HD2 PHE 14 -1.733 -3.185 0.367 1.00 0.00                            ATOM 236  HE1 PHE 14 1.396 -5.800 3.201 1.00 0.00                             ATOM 237  HE2 PHE 14 -0.539 -2.080 2.228 1.00 0.00                            ATOM 238  HZ PHE 14 1.053 -3.370 3.629 1.00 0.00                              ATOM 239  N GLY 15 -0.607 -7.896 -3.480 1.00 0.00                             ATOM 240  CA GLY 15 -0.995 -8.181 -4.849 1.00 0.00                            ATOM 241  C GLY 15 0.182 -8.794 -5.604 1.00 0.00                              ATOM 242  O GLY 15 1.159 -9.233 -4.999 1.00 0.00                              ATOM 243  H GLY 15 0.302 -8.231 -3.186 1.00 0.00                              ATOM 244 1HA GLY 15 -1.832 -8.881 -4.853 1.00 0.00                            ATOM 245 2HA GLY 15 -1.295 -7.254 -5.340 1.00 0.00                            ATOM 246  N NH2 16 0.095 -8.822 -6.935 1.00 0.00                              ATOM 247 1HN NH2 16 -0.720 -8.445 -7.397 1.00 0.00                            ATOM 248 2HN NH2 16 0.854 -9.209 -7.476 1.00 0.00                           __________________________________________________________________________

Example 9 Displacement of IGF-I from IGFBPs using (Leu²⁴, Ala³¹)hIGF

This example shows that the IGF mutant (Leu²⁴,Ala³¹)hIGF used in theabove animal studies can displace IGF-I tracer from IGFBPs present inhuman serum.

To determine if the IGF mutant could displace IGF-I from endogenousIGFBPs present in human serum, the following experiment was performed.Radiolabeled rhIGF-I (¹²⁵ I-IGF-I) was incubated with normal human serumsamples at a concentration of 33 ng/ml for 16 hr at 4° C. to allow forthe formation of ¹²⁵ I-IGF-I:IGFBP complexes. Following complexformation, the IGF mutant (Leu²⁴,Ala³¹)hIGF (40 μg/ml) was added to theincubations for 0, 0.5, 3, or 16 hr. The samples (n=3) were thenanalyzed by FPLC size-exclusion chromatography (FPLC-SEC) to determinethe relative size and amounts of the ¹²⁵ I-IGF-I:IGFBP complexes.

FIG. 41 is a chromatogram demonstrating the ability of the IGF mutant todisplace ¹²⁵ I-IGF-I from endogenous IGFBPs present in serum from normalhumans. Data are expressed as cpm per fraction (n=1). This experimentwas repeated three times, was quantitated, and is presented in Table IX.

                  TABLE IX                                                        ______________________________________                                        Quantitation of FPLC-SEC Data Demonstrating                                     Ability of IGF Mutant to Displace .sup.125 I-IGF-I                            From Endogenous IGFBPs in Normal Human Serum*                                          ˜150 kDa                                                                           ˜44 kDa                                                                           Free (˜7 kDa)                           ______________________________________                                        IGF Mutant 30.3 ± 1.4                                                                            60.5 ± 0.8                                                                            9.3 ± 2.3                                   (0 hr)                                                                        IGF Mutant 24.4 ± 0.8 37.3 ± 2.9 38.2 ± 3.7                          (0.5 hr)                                                                      IGF Mutant 10.3 ± 1.1 27.8 ± 1.5 61.9 ± 2.6                          (3 hr)                                                                        IGF Mutant  5.5 ± 0.1 12.6 ± 0.4 81.9 ± 0.5                          (24 hr)                                                                     ______________________________________                                         *Data (mean ± SD, n = 3) are expressed as percent of total                 radioactivity, calculated by dividing the amount of radioactivity in each     molecular weight range by total radioactivity in all three molecular          weight ranges.                                                           

After incubation of normal human serum with ¹²⁵ I-IGF-I for 16 hr, threepeaks of radioactivity were observed following FPLC-SEC (FIG. 42). Thesepeaks likely correspond to the following 125I-IGF-I complexes: ˜150 kDa(125I-IGF-I:IGFBP-3:ALS); ˜44 kDa (125I-IGF-I:IGFBPs 1-4); ˜7 kDa (free¹²⁵ I-IGF-I). As can be seen in FIG. 42, addition of the IGF mutant tothe serum resulted in a time-dependent decrease in ¹²⁵ I-IGF-Iassociated with the ¹²⁵ I-IGF-I:IGFBP complexes and an increase in theamount of free ¹²⁵ 1I-IGF-I. These results are outlined in the abovetable. The ¹²⁵ -I-IGF-I was more readily displaced from the ˜44 kDa thanthe ˜150 kDa ¹²⁵ I-IGF-I:IGFBP complex, suggesting that IGF-I bound tothe lower molecular weight IGFBPs is in a more bioavailable form. Thesedata clearly indicate that the IGF mutant has the ability to displaceIGF-I from endogenous IGFBPs present in normal human serum and thereforeis likely to be active in vivo in humans.

In conclusion, this evidence shows that one would expect the moleculesof the nature of the mutant to demonstrate the activities in humans thathave been shown in rats.

Example 10 Displacement of IGF-I from IGFBPs using BP15

This Example tests an IGFBP-3-specific peptide, BP15, for its ability toblock the binding of ¹²⁵ I-IGF-I in human serum. Human serum wasincubated with ¹²⁵ 1-IGF-I ± the peptide and the amount of tracer boundto IGFBPs via size-exclusion chromatography was measured. Addition ofthe peptide resulted in an approximately 42% decrease in ¹²⁵ I-IGF-Iassociated with the 150-KD IGF/IGFBP-3/ALS complex and a 59% increase inthe amount of free ¹²⁵ I-IGF-I. The peptide did not decrease ¹²⁵ I-IGF-Ibinding to the 44-KD IGFBPs (in fact, it slightly increased it),indicating that the peptide only competes with IGF-I for binding toIGFBP-3.

These results indicate that the analog (at 0.2 mM) can compete withIGF-I for binding to IGFBP-3 in human serum.

Example 11 Treatment of Humans with Human IGF-I

This example shows the principle of how an exogenously administeredcompound that binds to one or more of the IGFBPs acts to displaceendogenous IGFs and how to dose an IGF agonist for use in humans. Inthis study human Type II diabetics were administered recombinant humanIGF-I or placebo by twice-daily injection at four doses (10, 20, 40, or80 μg/kg) for 12 weeks. Blood samples were drawn, before, every twoweeks during, and after (EP) the 12 weeks of treatment. Theconcentrations of IGF-I, IGF-II, and IGFBP-3 were measured in all thesamples, with the exception of IGF-II not being measured in the samplestaken from the patients treated with 10 μg/day of IGF-I.

FIG. 43 shows the concentrations of IGF-I in the blood of the patients.The unexpected finding was the "plateau" effect of administering 40 and80 μg of IGF-I; the same total blood concentration of IGF-I was reachedwith these two doses. FIG. 44 shows the concentrations of IGF-II in theblood of the patients. In contrast to the rising levels of IGF-I, thelevels of IGF-II fell in almost a mirror-image pattern to the rise inIGF-I concentrations. As with the plateauing of the rising IGF-Iconcentrations, the falling IGF-II concentrations also reached aplateau.

FIG. 45 shows the concentrations of IGFBP-3 in the blood of thepatients. In contrast to the clear changes in the patterns of IGF-I andIGF-II in the blood, the concentrations of IGFBP-3 showed nostatistically significant or clear pattern of change.

Inspection of FIGS. 43 and 44 reveals that the total IGF concentrations(IGF-I plus IGF-II) showed little change with treatment. This wasbecause the rise in the concentrations of IGF-I closely matched the fallin the concentrations of IGF-II. Inspection of all three Figures showsthat the dose-related changes in the concentrations of IGF-I and IGF-IIin the blood of the patients were not accompanied by a reduced IGFBP-3binding protein capacity (IGFBP-3 is the major binding protein inblood).

The obvious explanation for the fall in the concentration of IGF-II, andthe plateauing of IGF-I and IGF-II concentrations, is that there is afinite amount of IGF binding protein capacity, and in this experimentthe doses of IGF-I used caused a dose-related displacement of IGF-IIfrom the binding proteins.

It is a logical extension of the observations in this Example to expectthat any molecule with the ability to enhance levels of active IGF wouldshow activities similar to those shown for IGF-I in this Example. Inaddition, from the doses of IGF-I used and the concentrations of IGFBPand IGF-I and IGF-II demonstrated, it is simple to calculate how much ofan IGF agonist should be given to increase levels of active endogenousIGF. The molar size relative to IGF-I, the affinity of the IGF agonistfor the IGFBP, and its bioavailability would be other variables takeninto account to arrive at doses that increased active IGF in a human.

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the objectives of the presentinvention.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 79                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Glu Leu Asp Gly Trp Val Cys Ile Lys Val - #Gly Glu Gln Asn Leu            1               - #5                  - #10                  - #15          - -  Cys Tyr Leu Ala Glu Gly                                                                   - #20  21                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  Trp Phe Lys Thr Val Cys Tyr Glu Trp Glu - #Asp Glu Val Gln Cys             1               - #5                  - #10                  - #15          - -  Tyr Thr Leu Glu Glu Gly                                                                   - #20  21                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Arg Val Gly Ala Tyr Ile Ser Cys Ser Glu - #Thr Glu Cys Trp Val             1               - #5                  - #10                  - #15          - -  Glu Asp Leu Leu Asp Gly                                                                   - #20  21                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -  Val Ala Trp Glu Val Cys Trp Asp Arg His - #Asp Gln Gly Tyr Ile             1               - #5                  - #10                  - #15          - -  Cys Thr Thr Asp Ser                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -  Ala Trp Glu Val Cys Trp Asp Arg His Gln - #Gly Tyr Ile Cys Thr             1               - #5                  - #10                  - #15          - -  Thr Asp Ser                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -  Cys Trp Asp Arg His Asp Gln Gly Tyr Ile - #Cys Thr Thr Asp Ser             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -  Glu Glu Ser Glu Cys Phe Glu Gly Pro Gly - #Tyr Val Ile Cys Gly             1               - #5                  - #10                  - #15          - -  Leu Val Gly                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -  Asp Met Gly Val Cys Ala Asp Gly Pro Trp - #Met Tyr Val Cys Glu             1               - #5                  - #10                  - #15          - -  Trp Thr Glu                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -  Ser Glu Glu Val Cys Trp Pro Val Ala Glu - #Trp Tyr Leu Cys Asn             1               - #5                  - #10                  - #15          - -  Met Trp Gly                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -  Ser Glu Glu Val Cys Trp Pro Val Ala Glu - #Trp Tyr Leu Cys Asn             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - -  Val Cys Trp Pro Val Ala Glu Trp Tyr Leu - #Cys Asn Met Trp Gly             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - -  Val Cys Trp Pro Val Ala Glu Trp Tyr Leu - #Cys Asn                         1               - #5                  - #10      12                         - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - -  Thr Gly Val Asp Cys Gln Cys Gly Pro Val - #His Cys Val Cys Met             1               - #5                  - #10                  - #15          - -  Asp Trp Ala                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - -  Thr Val Ala Asn Cys Asp Cys Tyr Met Pro - #Leu Cys Leu Cys Tyr             1               - #5                  - #10                  - #15          - -  Asp Ser Asp                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - -  Cys Arg Ala Gly Pro Leu Gln Trp Leu Cys - #Glu Lys Tyr Phe Gly             1               - #5                  - #10                  - #15          - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - -  Ser Glu Val Gly Cys Arg Ala Gly Pro Leu - #Gln Trp Leu Cys Glu             1               - #5                  - #10                  - #15          - -  Lys Tyr Phe Gly                                                                       19                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 400 base - #pairs                                                 (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - -  TCACGTAAAA AGGGTATCTA GAATTATGAT GATTACTCTG CGCAAACTTC - #                  50                                                                         - -  CTCTGGCGGT TGCCGTCGCA GCGGGCGTAA TGTCTGCTCA GGCCATGGCC - #                 100                                                                         - -  GGTCCCGAAA CTCTGTGCGG TGCTGAACTG GTTGACGCTC TGCAGTTCGT - #                 150                                                                         - -  ATGTGGTGAT CGAGGCTTCC TGTTCAACAA ACCGACTGGG GCTGGATCCT - #                 200                                                                         - -  CCTCTCGTCG TGCTCCCCAG ACTGGTATTG TTGACGAATG CTGCTTTCGT - #                 250                                                                         - -  TCTTGCGACC TGCGTCGTCT GGAAATGTAT TGCGCTCCCC TGAAACCCGC - #                 300                                                                         - -  TAAATCTGCT TAGAAGCTCC TAACGCTCGG TTGCCGCCGG GCGTTTTTTA - #                 350                                                                         - -  TTGTTAACTC ATGTTTGACA GCTTATCATC GATAAGCTTT AATGCGGTAG - #                 400                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 95 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - -  Met Met Ile Thr Leu Arg Lys Leu Pro Leu - #Ala Val Ala Val Ala             1               - #5                  - #10                  - #15          - -  Ala Gly Val Met Ser Ala Gln Ala Met Ala - #Gly Pro Glu Thr Leu                            - #20                  - #25                  - #30          - -  Cys Gly Ala Glu Leu Val Asp Ala Leu Gln - #Phe Val Cys Gly Asp                            - #35                  - #40                  - #45          - -  Arg Gly Phe Leu Phe Asn Lys Pro Thr Gly - #Ala Gly Ser Ser Ser                            - #50                  - #55                  - #60          - -  Arg Arg Ala Pro Gln Thr Gly Ile Val Asp - #Glu Cys Cys Phe Arg                            - #65                  - #70                  - #75          - -  Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr - #Cys Ala Pro Leu Lys                            - #80                  - #85                  - #90          - -  Pro Ala Lys Ser Ala                                                                       - #95                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5115 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Double                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - -  GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC - #                  50                                                                         - -  TCATTGCTGA GTTGTTATTT AAGCTTGCCC AAAAAGAAGA AGAGTCGAAT - #                 100                                                                         - -  GAACTGTGTG CGCAGGTAGA AGCTTTGGAG ATTATCGTCA CTGCAATGCT - #                 150                                                                         - -  TCGCAATATG GCGCAAAATG ACCAACAGCG GTTGATTGAT CAGGTAGAGG - #                 200                                                                         - -  GGGCGCTGTA CGAGGTAAAG CCCGATGCCA GCATTCCTGA CGACGATACG - #                 250                                                                         - -  GAGCTGCTGC GCGATTACGT AAAGAAGTTA TTGAAGCATC CTCGTCAGTA - #                 300                                                                         - -  AAAAGTTAAT CTTTTCAACA GCTGTCATAA AGTTGTCACG GCCGAGACTT - #                 350                                                                         - -  ATAGTCGCTT TGTTTTTATT TTTTAATGTA TTTGTAACTA GTACGCAAGT - #                 400                                                                         - -  TCACGTAAAA AGGGTATCTA GAATTATGAT GATTACTCTG CGCAAACTTC - #                 450                                                                         - -  CTCTGGCGGT TGCCGTCGCA GCGGGCGTAA TGTCTGCTCA GGCCATGGCC - #                 500                                                                         - -  GGTCCCGAAA CTCTGTGCGG TGCTGAACTG GTTGACGCTC TGCAGTTCGT - #                 550                                                                         - -  ATGTGGTGAT CGAGGCTTCC TGTTCAACAA ACCGACTGGG GCTGGATCCT - #                 600                                                                         - -  CCTCTCGTCG TGCTCCCCAG ACTGGTATTG TTGACGAATG CTGCTTTCGT - #                 650                                                                         - -  TCTTGCGACC TGCGTCGTCT GGAAATGTAT TGCGCTCCCC TGAAACCCGC - #                 700                                                                         - -  TAAATCTGCT TAGAAGCTCC TAACGCTCGG TTGCCGCCGG GCGTTTTTTA - #                 750                                                                         - -  TTGTTAACTC ATGTTTGACA GCTTATCATC GATAAGCTTT AATGCGGTAG - #                 800                                                                         - -  TTTATCACAG TTAAATTGCT AACGCAGTCA GGCACCGTGT ATGAAATCTA - #                 850                                                                         - -  ACAATGCGCT CATCGTCATC CTCGGCACCG TCACCCTGGA TGCTGTAGGC - #                 900                                                                         - -  ATAGGCTTGG TTATGCCGGT ACTGCCGGGC CTCTTGCGGG ATATCGTCCA - #                 950                                                                         - -  TTCCGACAGC ATCGCCAGTC ACTATGGCGT GCTGCTAGCG CTATATGCGT - #                1000                                                                         - -  TGATGCAATT TCTATGCGCA CCCGTTCTCG GAGCACTGTC CGACCGCTTT - #                1050                                                                         - -  GGCCGCCGCC CAGTCCTGCT CGCTTCGCTA CTTGGAGCCA CTATCGACTA - #                1100                                                                         - -  CGCGATCATG GCGACCACAC CCGTCCTGTG GATCCTCTAC GCCGGACGCA - #                1150                                                                         - -  TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG CGCCTATATC - #                1200                                                                         - -  GCCGACATCA CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG - #                1250                                                                         - -  CGCTTGTTTC GGCGTGGGTA TGGTGGCAGG CCCCGTGGCC GGGGGACTGT - #                1300                                                                         - -  TGGGCGCCAT CTCCTTGCAT GCACCATTCC TTGCGGCGGC GGTGCTCAAC - #                1350                                                                         - -  GGCCTCAACC TACTACTGGG CTGCTTCCTA ATGCAGGAGT CGCATAAGGG - #                1400                                                                         - -  AGAGCGTCGA CCGATGCCCT TGAGAGCCTT CAACCCAGTC AGCTCCTTCC - #                1450                                                                         - -  GGTGGGCGCG GGGCATGACT ATCGTCGCCG CACTTATGAC TGTCTTCTTT - #                1500                                                                         - -  ATCATGCAAC TCGTAGGACA GGTGCCGGCA GCGCTCTGGG TCATTTTCGG - #                1550                                                                         - -  CGAGGACCGC TTTCGCTGGA GCGCGACGAT GATCGGCCTG TCGCTTGCGG - #                1600                                                                         - -  TATTCGGAAT CTTGCACGCC CTCGCTCAAG CCTTCGTCAC TGGTCCCGCC - #                1650                                                                         - -  ACCAAACGTT TCGGCGAGAA GCAGGCCATT ATCGCCGGCA TGGCGGCCGA - #                1700                                                                         - -  CGCGCTGGGC TACGTCTTGC TGGCGTTCGC GACGCGAGGC TGGATGGCCT - #                1750                                                                         - -  TCCCCATTAT GATTCTTCTC GCTTCCGGCG GCATCGGGAT GCCCGCGTTG - #                1800                                                                         - -  CAGGCCATGC TGTCCAGGCA GGTAGATGAC GACCATCAGG GACAGCTTCA - #                1850                                                                         - -  AGGATCGCTC GCGGCTCTTA CCAGCCTAAC TTCGATCACT GGACCGCTGA - #                1900                                                                         - -  TCGTCACGGC GATTTATGCC GCCTCGGCGA GCACATGGAA CGGGTTGGCA - #                1950                                                                         - -  TGGATTGTAG GCGCCGCCCT ATACCTTGTC TGCCTCCCCG CGTTGCGTCG - #                2000                                                                         - -  CGGTGCATGG AGCCGGGCCA CCTCGACCTG AATGGAAGCC GGCGGCACCT - #                2050                                                                         - -  CGCTAACGGA TTCACCACTC CAAGAATTGG AGCCAATCAA TTCTTGCGGA - #                2100                                                                         - -  GAACTGTGAA TGCGCAAACC AACCCTTGGC AGAACATATC CATCGCGTCC - #                2150                                                                         - -  GCCATCTCCA GCAGCCGCAC GCGGCGCATC TCGGGCAGCG TTGGGTCCTG - #                2200                                                                         - -  GCCACGGGTG CGCATGATCG TGCTCCTGTC GTTGAGGACC CGGCTAGGCT - #                2250                                                                         - -  GGCGGGGTTG CCTTACTGGT TAGCAGAATG AATCACCGAT ACGCGAGCGA - #                2300                                                                         - -  ACGTGAAGCG ACTGCTGCTG CAAAACGTCT GCGACCTGAG CAACAACATG - #                2350                                                                         - -  AATGGTCTTC GGTTTCCGTG TTTCGTAAAG TCTGGAAACG CGGAAGTCAG - #                2400                                                                         - -  CGCCCTGCAC CATTATGTTC CGGATCTGCA TCGCAGGATG CTGCTGGCTA - #                2450                                                                         - -  CCCTGTGGAA CACCTACATC TGTATTAACG AAGCGCTGGC ATTGACCCTG - #                2500                                                                         - -  AGTGATTTTT CTCTGGTCCC GCCGCATCCA TACCGCCAGT TGTTTACCCT - #                2550                                                                         - -  CACAACGTTC CAGTAACCGG GCATGTTCAT CATCAGTAAC CCGTATCGTG - #                2600                                                                         - -  AGCATCCTCT CTCGTTTCAT CGGTATCATT ACCCCCATGA ACAGAAATTC - #                2650                                                                         - -  CCCCTTACAC GGAGGCATCA AGTGACCAAA CAGGAAAAAA CCGCCCTTAA - #                2700                                                                         - -  CATGGCCCGC TTTATCAGAA GCCAGACATT AACGCTTCTG GAGAAACTCA - #                2750                                                                         - -  ACGAGCTGGA CGCGGATGAA CAGGCAGACA TCTGTGAATC GCTTCACGAC - #                2800                                                                         - -  CACGCTGATG AGCTTTACCG CAGCTGCCTC GCGCGTTTCG GTGATGACGG - #                2850                                                                         - -  TGAAAACCTC TGACACATGC AGCTCCCGGA GACGGTCACA GCTTGTCTGT - #                2900                                                                         - -  AAGCGGATGC CGGGAGCAGA CAAGCCCGTC AGGGCGCGTC AGCGGGTGTT - #                2950                                                                         - -  GGCGGGTGTC GGGGCGCAGC CATGACCCAG TCACGTAGCG ATAGCGGAGT - #                3000                                                                         - -  GTATACTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA - #                3050                                                                         - -  CCATATGCGG TGTGAAATAC CGCACAGATG CGTAAGGAGA AAATACCGCA - #                3100                                                                         - -  TCAGGCGCTC TTCCGCTTCC TCGCTCACTG ACTCGCTGCG CTCGGTCGTT - #                3150                                                                         - -  CGGCTGCGGC GAGCGGTATC AGCTCACTCA AAGGCGGTAA TACGGTTATC - #                3200                                                                         - -  CACAGAATCA GGGGATAACG CAGGAAAGAA CATGTGAGCA AAAGGCCAGC - #                3250                                                                         - -  AAAAGGCCAG GAACCGTAAA AAGGCCGCGT TGCTGGCGTT TTTCCATAGG - #                3300                                                                         - -  CTCCGCCCCC CTGACGAGCA TCACAAAAAT CGACGCTCAA GTCAGAGGTG - #                3350                                                                         - -  GCGAAACCCG ACAGGACTAT AAAGATACCA GGCGTTTCCC CCTGGAAGCT - #                3400                                                                         - -  CCCTCGTGCG CTCTCCTGTT CCGACCCTGC CGCTTACCGG ATACCTGTCC - #                3450                                                                         - -  GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT TCTCATAGCT CACGCTGTAG - #                3500                                                                         - -  GTATCTCAGT TCGGTGTAGG TCGTTCGCTC CAAGCTGGGC TGTGTGCACG - #                3550                                                                         - -  AACCCCCCGT TCAGCCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT - #                3600                                                                         - -  GAGTCCAACC CGGTAAGACA CGACTTATCG CCACTGGCAG CAGCCACTGG - #                3650                                                                         - -  TAACAGGATT AGCAGAGCGA GGTATGTAGG CGGTGCTACA GAGTTCTTGA - #                3700                                                                         - -  AGTGGTGGCC TAACTACGGC TACACTAGAA GGACAGTATT TGGTATCTGC - #                3750                                                                         - -  GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA AGAGTTGGTA GCTCTTGATC - #                3800                                                                         - -  CGGCAAACAA ACCACCGCTG GTAGCGGTGG TTTTTTTGTT TGCAAGCAGC - #                3850                                                                         - -  AGATTACGCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT - #                3900                                                                         - -  ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT - #                3950                                                                         - -  CATGAGATTA TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT - #                4000                                                                         - -  GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT - #                4050                                                                         - -  TACCAATGCT TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG - #                4100                                                                         - -  TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT ACGATACGGG - #                4150                                                                         - -  AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC - #                4200                                                                         - -  TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA - #                4250                                                                         - -  GCGCAGAAGT GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT - #                4300                                                                         - -  GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC - #                4350                                                                         - -  GTTGTTGCCA TTGCTGCAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT - #                4400                                                                         - -  GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC - #                4450                                                                         - -  CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC - #                4500                                                                         - -  AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA - #                4550                                                                         - -  TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG - #                4600                                                                         - -  AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC - #                4650                                                                         - -  TCTTGCCCGG CGTCAACACG GGATAATACC GCGCCACATA GCAGAACTTT - #                4700                                                                         - -  AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA - #                4750                                                                         - -  TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC - #                4800                                                                         - -  TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC - #                4850                                                                         - -  AGGAAGGCAA AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT - #                4900                                                                         - -  GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT - #                4950                                                                         - -  TATTGTCTCA TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA - #                5000                                                                         - -  AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GACGTCTAAG - #                5050                                                                         - -  AAACCATTAT TATCATGACA TTAACCTATA AAAATAGGCG TATCACGAGG - #                5100                                                                         - -  CCCTTTCGTC TTCAA             - #                  - #                      - #  5115                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5140 base - #pairs                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - -  GAATTCAACT TCTCCATACT TTGGATAAGG AAATACAGAC ATGAAAAATC - #                  50                                                                         - -  TCATTGCTGA GTTGTTATTT AAGCTTGCCC AAAAAGAAGA AGAGTCGAAT - #                 100                                                                         - -  GAACTGTGTG CGCAGGTAGA AGCTTTGGAG ATTATCGTCA CTGCAATGCT - #                 150                                                                         - -  TCGCAATATG GCGCAAAATG ACCAACAGCG GTTGATTGAT CAGGTAGAGG - #                 200                                                                         - -  GGGCGCTGTA CGAGGTAAAG CCCGATGCCA GCATTCCTGA CGACGATACG - #                 250                                                                         - -  GAGCTGCTGC GCGATTACGT AAAGAAGTTA TTGAAGCATC CTCGTCAGTA - #                 300                                                                         - -  AAAAGTTAAT CTTTTCAACA GCTGTCATAA AGTTGTCACG GCCGAGACTT - #                 350                                                                         - -  ATAGTCGCTT TGTTTTTATT TTTTAATGTA TTTGTAACTA GTACGCAAGT - #                 400                                                                         - -  TCACGTAAAA AGGGTATCTA GAGGTTGAGG TGATTTTATG AAAAAGAATA - #                 450                                                                         - -  TCGCATTTCT TCTTGCATCT ATGTTCGTTT TTTCTATTGC TACAAATGCC - #                 500                                                                         - -  TATGCATCTG GTACCGCCAT GGCTGATCCG AACCGTTTCC GCGGTAAAGA - #                 550                                                                         - -  TCTGGCAGGT TCACCAGGTG GAGGATCCGG AGGAGGCGCC GAGGGTGACG - #                 600                                                                         - -  ATCCCGCAAA AGCGGCCTTT AACTCCCTGC AAGCCTCAGC GACCGAATAT - #                 650                                                                         - -  ATCGGTTATG CGTGGGCGAT GGTTGTTGTC ATTGTCGGCG CAACTATCGG - #                 700                                                                         - -  TATCAAGCTG TTTAAGAAAT TCACCTCGAA AGCAAGCTGA TAAACCGATA - #                 750                                                                         - -  CAATTAAAGG CTCCTTTTGG AGCCTTTTTT TTTGGAGATT TTCAACGTGA - #                 800                                                                         - -  AAAAATTATT ATTCGCAATT CCTTTAGTTG TTCCTTTCTA TTCTCACTCC - #                 850                                                                         - -  GCTGAAACTG TTGAAAGTTG TTTAGCAAAA CCCCATACAG AAAATTCATT - #                 900                                                                         - -  TACTAACGTC TGGAAAGACG ACAAAACTTT AGATCGTTAC GCTAACTATG - #                 950                                                                         - -  AGGGTTGTCT GTGGAATGCT ACAGGCGTTG TAGTTTGTAC TGGTGACGAA - #                1000                                                                         - -  ACTCAGTGTC TAGCTAGAGT GGCGGTGGCT CTGGTTCCGG TGATTTTGAT - #                1050                                                                         - -  TATGAAAAGA TGGCAAACGC TAATAAGGGG GCTATGACCG AAAATGCCGA - #                1100                                                                         - -  TGAAAACGCG CTACAGTCTG ACGCTAAAGG CAAACTTGAT TCTGTCGCTA - #                1150                                                                         - -  CTGATTACGG TGCTGCTATC GATGGTTTCA TTGGTGACGT TTCCGGCCTT - #                1200                                                                         - -  GCTAATGGTA ATGGTGCTAC TGGTGATTTT GCTGGCTCTA ATTCCCAAAT - #                1250                                                                         - -  GGCTCAAGTC GGTGACGGTG ATAATTCACC TTTAATGAAT AATTTCCGTC - #                1300                                                                         - -  AATATTTACC TTCCCTCCCT CAATCGGTTG AATGTCGCCC TTTTGTCTTT - #                1350                                                                         - -  AGCGCTGGTA AACCATATGA ATTTTCTATT GATTGTGACA AAATAAACTT - #                1400                                                                         - -  ATTCCGTGGT GTCTTTGCGT TTCTTTTATA TGTTGCCACC TTTATGTATG - #                1450                                                                         - -  TATTTTCTAC GTTTGCTAAC ATACTGCGTA ATAAGGAGTC TTAATCATGC - #                1500                                                                         - -  CAGTTCTTTT GGCTAGCGCC GCCCTATACC TTGTCTGCCT CCCCGCGTTG - #                1550                                                                         - -  CGTCGCGGTG CATGGAGCCG GGCCACCTCG ACCTGAATGG AAGCCGGCGG - #                1600                                                                         - -  CACCTCGCTA ACGGATTCAC CACTCCAAGA ATTGGAGCCA ATCAATTCTT - #                1650                                                                         - -  GCGGAGAACT GTGAATGCGC AAACCAACCC TTGGCAGAAC ATATCCATCG - #                1700                                                                         - -  CGTCCGCCAT CTCCAGCAGC CGCACGCGGC GCATCTCGGG CAGCGTTGGG - #                1750                                                                         - -  TCCTGGCCAC GGGTGCGCAT GATCGTGCTC CTGTCGTTGA GGACCCGGCT - #                1800                                                                         - -  AGGCTGGCGG GGTTGCCTTA CTGGTTAGCA GAATGAATCA CCGATACGCG - #                1850                                                                         - -  AGCGAACGTG AAGCGACTGC TGCTGCAAAA CGTCTGCGAC CTGAGCAACA - #                1900                                                                         - -  ACATGAATGG TCTTCGGTTT CCGTGTTTCG TAAAGTCTGG AAACGCGGAA - #                1950                                                                         - -  GTCAGCGCCC TGCACCATTA TGTTCCGGAT CTGCATCGCA GGATGCTGCT - #                2000                                                                         - -  GGCTACCCTG TGGAACACCT ACATCTGTAT TAACGAAGCG CTGGCATTGA - #                2050                                                                         - -  CCCTGAGTGA TTTTTCTCTG GTCCCGCCGC ATCCATACCG CCAGTTGTTT - #                2100                                                                         - -  ACCCTCACAA CGTTCCAGTA ACCGGGCATG TTCATCATCA GTAACCCGTA - #                2150                                                                         - -  TCGTGAGCAT CCTCTCTCGT TTCATCGGTA TCATTACCCC CATGAACAGA - #                2200                                                                         - -  AATTCCCCCT TACACGGAGG CATCAAGTGA CCAAACAGGA AAAAACCGCC - #                2250                                                                         - -  CTTAACATGG CCCGCTTTAT CAGAAGCCAG ACATTAACGC TTCTGGAGAA - #                2300                                                                         - -  ACTCAACGAG CTGGACGCGG ATGAACAGGC AGACATCTGT GAATCGCTTC - #                2350                                                                         - -  ACGACCACGC TGATGAGCTT TACCGCAGGA TCCGGAAATT GTAAACGTTA - #                2400                                                                         - -  ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT - #                2450                                                                         - -  AACCAATAGG CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC - #                2500                                                                         - -  CGAGATAGGG TTGAGTGTTG TTCCAGTTTG GAACAAGAGT CCACTATTAA - #                2550                                                                         - -  AGAACGTGGA CTCCAACGTC AAAGGGCGAA AAACCGTCTA TCAGGGCTAT - #                2600                                                                         - -  GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG GGTCGAGGTG - #                2650                                                                         - -  CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT - #                2700                                                                         - -  GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA - #                2750                                                                         - -  GGAGCGGGCG CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC - #                2800                                                                         - -  CACCACACCC GCCGCGCTTA ATGCGCCGCT ACAGGGCGCG TCCGGATCCT - #                2850                                                                         - -  GCCTCGCGCG TTTCGGTGAT GACGGTGAAA ACCTCTGACA CATGCAGCTC - #                2900                                                                         - -  CCGGAGACGG TCACAGCTTG TCTGTAAGCG GATGCCGGGA GCAGACAAGC - #                2950                                                                         - -  CCGTCAGGGC GCGTCAGCGG GTGTTGGCGG GTGTCGGGGC GCAGCCATGA - #                3000                                                                         - -  CCCAGTCACG TAGCGATAGC GGAGTGTATA CTGGCTTAAC TATGCGGCAT - #                3050                                                                         - -  CAGAGCAGAT TGTACTGAGA GTGCACCATA TGCGGTGTGA AATACCGCAC - #                3100                                                                         - -  AGATGCGTAA GGAGAAAATA CCGCATCAGG CGCTCTTCCG CTTCCTCGCT - #                3150                                                                         - -  CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG GTATCAGCTC - #                3200                                                                         - -  ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA - #                3250                                                                         - -  AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC - #                3300                                                                         - -  CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA - #                3350                                                                         - -  AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG ACTATAAAGA - #                3400                                                                         - -  TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC CTGTTCCGAC - #                3450                                                                         - -  CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG GGAAGCGTGG - #                3500                                                                         - -  CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT - #                3550                                                                         - -  CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG - #                3600                                                                         - -  CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT - #                3650                                                                         - -  TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG AGCGAGGTAT - #                3700                                                                         - -  GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT ACGGCTACAC - #                3750                                                                         - -  TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA GTTACCTTCG - #                3800                                                                         - -  GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC - #                3850                                                                         - -  GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC - #                3900                                                                         - -  TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG - #                3950                                                                         - -  AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA AAGGATCTTC - #                4000                                                                         - -  ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA TCTAAAGTAT - #                4050                                                                         - -  ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC AGTGAGGCAC - #                4100                                                                         - -  CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC - #                4150                                                                         - -  GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC - #                4200                                                                         - -  TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA - #                4250                                                                         - -  TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA - #                4300                                                                         - -  TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG - #                4350                                                                         - -  TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT GCAGGCATCG - #                4400                                                                         - -  TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA - #                4450                                                                         - -  CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG - #                4500                                                                         - -  CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT - #                4550                                                                         - -  CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC - #                4600                                                                         - -  GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA - #                4650                                                                         - -  ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ACACGGGATA - #                4700                                                                         - -  ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT - #                4750                                                                         - -  TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC - #                4800                                                                         - -  GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA - #                4850                                                                         - -  CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG - #                4900                                                                         - -  GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATACTCT TCCTTTTTCA - #                4950                                                                         - -  ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT - #                5000                                                                         - -  TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC - #                5050                                                                         - -  CGAAAAGTGC CACCTGACGT CTAAGAAACC ATTATTATCA TGACATTAAC - #                5100                                                                         - -  CTATAAAAAT AGGCGTATCA CGAGGCCCTT TCGTCTTCAA    - #                      - #  5140                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 77 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - -  Ser Gly Thr Ala Met Ala Asp Pro Asn Arg - #Phe Arg Gly Lys Asp             1               - #5                  - #10                  - #15          - -  Leu Ala Gly Ser Pro Gly Gly Gly Ser Gly - #Gly Gly Ala Glu Gly                            - #20                  - #25                  - #30          - -  Asp Asp Pro Ala Lys Ala Ala Phe Asn Ser - #Leu Gln Ala Ser Ala                            - #35                  - #40                  - #45          - -  Thr Glu Tyr Ile Gly Tyr Ala Trp Ala Met - #Val Val Val Ile Val                            - #50                  - #55                  - #60          - -  Gly Ala Thr Ile Gly Ile Lys Leu Phe Lys - #Lys Phe Thr Ser Lys                            - #65                  - #70                  - #75          - -  Ala Ser                                                                       77                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - -  GTTCGTATGT GGTGATCGAG GCTTCCTGTT CAACAAACCG ACTGGGGCTG - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (ii) MOLECULE TYPE: Nucleic Acid                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - -  GATCCAGCCC CAGTCGGTTT GTTGAACAGG AAGCCTCGAT CACCACATAC - #                  50                                                                         - -  GAACTGCA               - #                  - #                  -     #          58                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - -  Ser Gly Thr Ala Cys Xaa Xaa Gly Pro Xaa - #Xaa Xaa Xaa Cys Ser             1               - #5                  - #10                  - #15          - -  Leu Ala Gly Ser Pro                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - -  Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Pro Xaa - #Xaa Xaa Xaa Cys Xaa             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - -  Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa - #Xaa Xaa Xaa Xaa Xaa             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - -  Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa - #Xaa Xaa Cys Xaa Xaa             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - -  Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa - #Xaa Xaa Xaa Cys Xaa             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - -  Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa - #Xaa Xaa Xaa Cys Xaa             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - -  Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa - #Xaa Xaa Xaa Xaa Cys             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - -  Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa - #Xaa Xaa Xaa Xaa Cys             1               - #5                  - #10                  - #15          - -  Xaa Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - -  Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa - #Xaa Xaa Xaa Xaa Xaa             1               - #5                  - #10                  - #15          - -  Cys Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - -  Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa - #Xaa Xaa Xaa Xaa Xaa             1               - #5                  - #10                  - #15          - -  Cys Xaa Xaa Xaa Xaa                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - -  Gly Gly Thr Tyr Ser Cys His Phe Gly Pro - #Leu Thr Trp Val Cys             1               - #5                  - #10                  - #15          - -  Lys Pro Gln Gly Gly                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - -  Cys Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Cys                                    1               - #5                  - #10                                 - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - -  GCCTATGCAT CTGGTACCGC CTGCNNSNNS GGTCCTNNSN NSNNSNNSTG - #                  50                                                                         - -  TTCTCTGGCA GGTTCACCAG           - #                  - #                      - # 70                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (ii) MOLECULE TYPE: Nucleic Acid                                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS TGCNNSNNSG GTCCTNNSNN - #                  50                                                                         - -  SNNSNNSTGT NNSNNSNNSN NSGGTGGAGG ATCCGGAGGA G   - #                      - #   91                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSNNSNNST GCNNSNNSNN - #                  50                                                                         - -  SNNSTGCNNS NNSNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSNNSNNST GCNNSNNSNN - #                  50                                                                         - -  SNNSNNSTGC NNSNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSNNSTGCN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSTGC NNSNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSNNSTGCN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSNNS TGCNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSTGCNNSN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSNNS TGCNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSTGCNNSN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSNNS NNSTGCNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS TGCNNSNNSN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSNNS NNSTGCNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base - #pairs                                                  (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - -  GCTACAAATG CCTATGCANN SNNSNNSNNS NNSNNSNNSN NSNNSNNSNN - #                  50                                                                         - -  SNNSNNSNNS NNSNNSNNSN NSNNSNNSGG TGGAGGATCC GGAGGAG  - #                    97                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - -  Ser Gly Thr Ala Cys Tyr Gly Gly Pro Glu - #Trp Trp Cys Cys Ser             1               - #5                  - #10                  - #15          - -  Leu Ala Gly Ser Pro                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - -  Asp Leu Ala Ile Cys Ala Glu Gly Pro Glu - #Ile Trp Val Cys Glu             1               - #5                  - #10                  - #15          - -  Glu Thr Ser                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - -  Asp Phe Trp Ile Cys Leu Ser Gly Pro Gly - #Trp Glu Glu Cys Leu             1               - #5                  - #10                  - #15          - -  Glu Trp Trp                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - -  Gly Ser Ala Gly Gln Gly Met Thr Glu Glu - #Trp Ala Trp Ile Trp             1               - #5                  - #10                  - #15          - -  Glu Trp Trp Lys Glu                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - -  Glu Leu Asp Gly Trp Val Cys Ile Lys Val - #Gly Glu Gln Asn Leu             1               - #5                  - #10                  - #15          - -  Cys Tyr Leu Ala Glu                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - -  Ala Ile Gly Gly Trp Cys Phe Ile Glu Leu - #Asp Ser Leu Trp Cys             1               - #5                  - #10                  - #15          - -  Glu Glu Gln Ile Gly                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - -  Ser Glu Asp Val Glu Cys Trp Gln Val Trp - #Glu Asn Leu Val Cys             1               - #5                  - #10                  - #15          - -  Ser Val Glu His Arg                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - -  Ser Glu Glu Val Cys Trp Pro Val Ala Glu - #Trp Tyr Leu Cys Asn             1               - #5                  - #10                  - #15          - -  Met Trp Gly Arg                                                                       19                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - -  Arg Val Gly Ala Tyr Ile Ser Cys Ser Glu - #Thr Glu Cys Trp Val             1               - #5                  - #10                  - #15          - -  Glu Asp Leu Leu Asp                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - -  Trp Phe Lys Thr Val Cys Tyr Glu Trp Glu - #Asp Glu Val Gln Cys             1               - #5                  - #10                  - #15          - -  Tyr Thr Leu Glu Glu                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - -  Arg Leu Glu Glu Gln Cys Val Glu Val Asn - #Tyr Glu Pro Ser Cys             1               - #5                  - #10                  - #15          - -  Ser Phe Thr Ala Asn                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - -  Ser Glu Glu Val Cys Trp Pro Val Ala Glu - #Trp Tyr Leu Cys Asn             1               - #5                  - #10                  - #15          - -  Ile Leu Gly Pro                                                                       19                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - -  Glu Thr Val Ala Asn Cys Asp Cys Tyr Met - #Asp Leu Cys Leu Cys             1               - #5                  - #10                  - #15          - -  Tyr Gly Ser Asp Arg                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - -  Tyr His Pro Ile Ser Cys Met Asp His Tyr - #Tyr Leu Ile Ile Cys             1               - #5                  - #10                  - #15          - -  Asp Glu Thr Val Asn                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - -  Ala Glu Trp Ala Glu Cys Trp Ile Ala Gly - #Asp Gln Leu Leu Cys             1               - #5                  - #10                  - #15          - -  Val Gly Lys Asp Asn                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - -  Glu Pro Trp Leu Cys Gln Tyr Tyr Glu Ala - #Ala Met Leu Tyr Leu             1               - #5                  - #10                  - #15          - -  Cys Trp Glu Glu Gly                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - -  Ala Glu Glu Gly Met Val Trp Gly Trp Thr - #Gly Gly Trp Tyr Asn             1               - #5                  - #10                  - #15          - -  Leu Asp Glu Leu Cys                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - -  Ser Gly Gly Ala Ile Tyr Trp Pro Val Glu - #Gln Phe Ile Ala Phe             1               - #5                  - #10                  - #15          - -  Met Ala Val Gly Lys                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - -  Ser Gly Gly Ala Ile Tyr Met Pro Val Glu - #Gln Phe Ile Ala Phe             1               - #5                  - #10                  - #15          - -  Met Ala Val Gly Lys                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - -  Glu Val Leu Leu Cys Ser Asp Gly Pro Gln - #Leu Tyr Leu Cys Glu             1               - #5                  - #10                  - #15          - -  Leu Tyr Ala                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - -  Ser Gly Val Glu Cys Val Trp Gly Pro Gln - #Trp Gly Phe Cys Val             1               - #5                  - #10                  - #15          - -  Glu Glu Tyr                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - -  Asp Lys Glu Val Cys Tyr Leu Gly Pro Glu - #Thr Trp Leu Cys Phe             1               - #5                  - #10                  - #15          - -  Trp Trp Pro                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - -  Gly Asp Val Glu Cys Ile Glu Gly Pro Trp - #Gly Glu Leu Cys Val             1               - #5                  - #10                  - #15          - -  Trp Ala Asp                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - -  Phe Gly Gly Trp Ser Cys Gln Pro Thr Trp - #Val Asp Val Tyr Val             1               - #5                  - #10                  - #15          - -  Cys Asn Phe Glu Glu                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - -  Ala Met Trp Val Cys Val Ser Asp Trp Glu - #Thr Val Glu Glu Cys             1               - #5                  - #10                  - #15          - -  Ile Gln Tyr Met Tyr                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - -  Thr Asn Trp Phe Phe Val Cys Glu Ser Gly - #His Gln Asp Ile Cys             1               - #5                  - #10                  - #15          - -  Trp Leu Ala Glu Glu                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - -  Ser Glu Val Gly Cys Arg Ala Gly Pro Leu - #Gln Leu Cys Glu Lys             1               - #5                  - #10                  - #15          - -  Tyr Phe                                                                       17                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - -  Lys Asp Pro Val Cys Gly Glu Gly Pro Leu - #Met Arg Ile Cys Glu             1               - #5                  - #10                  - #15          - -  Arg Leu Phe Gly                                                                       19                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - -  Glu Val Asp Gly Arg Trp Trp Ile Val Glu - #Thr Phe Leu Ala Lys             1               - #5                  - #10                  - #15          - -  Trp Asp His Met Ala Gly                                                                   - #20  21                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - -  Trp Val Met Glu Cys Gly Ala Gly Pro Trp - #Pro Glu Gly Cys Thr             1               - #5                  - #10                  - #15          - -  Phe Met Leu                                                                       18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - -  Arg Lys Thr Ser Gln Gly Arg Gly Gln Glu - #Met Cys Trp Glu Thr             1               - #5                  - #10                  - #15          - -  Gly Gly Cys Ser                                                                       19                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - -  Ser Trp Glu Arg Gly Glu Leu Thr Tyr Met - #Lys Leu Cys Glu Tyr             1               - #5                  - #10                  - #15          - -  Met Arg Leu Gln Gln                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - -  Glu His Gly Arg Ala Asn Cys Leu Ile Thr - #Pro Glu Ala Gly Lys             1               - #5                  - #10                  - #15          - -  Leu Ala Arg Val Thr                                                                       - #20                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - -  Val Glu Asp Glu Cys Trp Met Gly Pro Asp - #Trp Ala Val Cys Trp             1               - #5                  - #10                  - #15          - -  Thr Trp Gly                                                                       18                                                                 __________________________________________________________________________

What is claimed is:
 1. A peptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, and SEQ ID NO:16.
 2. The peptide of claim 1comprising an amino acid sequence selected from the group consisting ofSEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. 3.The peptide of claim 1 comprising an amino acid sequence that is SEQ IDNO:4, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:16.
 4. The peptide of claim 1 comprising an aminoacid sequence that is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 15, or SEQID NO:
 16. 5. The peptide of claim 1 comprising an amino acid sequencethat is SEQ ID NO: 15 or SEQ ID NO:16.
 6. A composition comprising thepeptide of claim 1 in a carrier.
 7. The composition of claim 6 that issterile.
 8. The composition of claim 6 further comprising a growthhormone, a growth hormone releasing peptide, a growth hormone releasinghormone, a growth hormone secretagogue, an IGF, an IGF in combinationwith an IGF binding protein, an IGF binding protein, growth hormone incombination with growth hormone binding protein, insulin, athiazolidinedione, or a hypoglycemic agent.
 9. A peptide comprising anamino acid sequence that is SEQ ID NO:10, SEQ ID NO:15, or SEQ ID NO:16.